Wnt receptor compositions and methods

ABSTRACT

Wnt receptor compositions and methods of use are disclosed. In particular, methods using Wnt receptors, such as Dfz2, in screens for compounds which modulate the binding of a Wnt polypeptide to a Wnt receptor.

[0001] The present invention claims priority under 35 USC 120 to U.S. provisional patent application No. 60/015,307.

[0002] This work was supported in part by a grant from the National Cancer Institute. Accordingly, the United States Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to Wnt receptor compositions and therapeutic and diagnostic methods related thereto.

[0004] References

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BACKGROUND OF THE INVENTION

[0051] Wnt genes encode secreted proteins involved in cell-to-cell signaling. Wnt genes play important growth controlling roles, in particular in the mammary gland, and act as oncogenes in mouse mammary tumors. Little is known about the mechanism of action of Wnt products, in part because Wnt receptors have until now remained unidentified.

SUMMARY OF THE INVENTION

[0052] In one aspect, the present invention includes an isolated nucleic acid molecule encoding a Wnt receptor (WntR) polypeptide. In a general embodiment, the WntR polypeptide has an amino acid sequence that is greater than about 90% identical to the amino acid sequence of at least one of the following Wnt receptors: Dfz1, Dfz2, Rfz1, Rfz2, Hfz3, Hfz4, Hfz5, Mfz3, Mfz4, Mfz5, Mfz6, Mfz7, Mfz8, and Cfz1. In a related embodiment, the Wnt receptor has an amino acid sequence that is more than about 90% identical to an amino acid sequence contained SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16. Exemplary WntRs described herein have an amino acid sequence that is more than about 90% identical to one of the following sequences: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16. In preferred embodiments, the WntR polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0053] Examples of nucleic acid molecules encoding Wnt receptor polypeptides are provided herein as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15. Preferred embodiments are human Wnt polynucleotides. An exemplary human Wnt polynucleotide has the sequence presented as SEQ ID NO:9.

[0054] The invention further includes fragments of polynucleotides encoding full-length WntR, where the fragments are of sufficient length to hybridize selectively with a Wnt polynucleotide sequence or complement thereof, such as a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15. Such fragments are at least 15, preferably at least about 18, 21 or 24, nucleotides in length.

[0055] In another aspect, the invention includes an isolated Wnt receptor polypeptide. In a general embodiment, the polypeptide has an amino acid sequence that is more than about 90% identical to the amino acid sequence of a Wnt receptor selected from the group consisting of Dfz1, Dfz2, Rfz1, Rfz2, Hfz3, Hfz4, Hfz5, Mfz3, Mfz4, Mfz5, Mfz6, Mfz7, Mfz8, and Cfz1. In a related embodiment, the polypeptide has an amino acid sequence that is more than about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16. In another related embodiment, the polypeptide sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0056] Preferred embodiments are human Wnt polypeptides. An exemplary human Wnt polypeptide has the sequence presented as SEQ ID NO:10.

[0057] The invention further includes peptide fragments derived from a full-length WntR polypeptide, where the fragments contain a region of at least seven, preferably at least ten, consecutive amino acids, and where the region has at least about an 80% identity with the residues of a corresponding region of a polypeptide having a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0058] Also included in the invention are antibodies, both monoclonal and polyclonal, specifically-immunoreactive with Wnt receptor polypeptides. Such antibodies may be produced using standard methods (Harlow).

[0059] The invention also includes a method of identifying a compound capable of affecting binding of a Wnt polypeptide to a Wnt receptor polypeptide. The method includes (i) contacting such a Wnt receptor polypeptide with a selected Wnt polypeptide, in the presence and absence of a test compound, (ii) measuring the effect of the test compound on the extent of binding between the Wnt polypeptide and the Wnt receptor polypeptide, and (iii) identifying said compound as effective if its measured effect on the extent of binding is above a threshold level. In a general embodiment, the method includes an additional step (iv) comprising preparing a pharmaceutical preparation of a compound identified as effective to alter binding of a Wnt polypeptide to a WntR polypeptide.

[0060] In one embodiment, the threshold is a 2-fold or greater inhibition of binding. In another embodiment, the threshold is a 2-fold or greater potentiation of binding. Examples of suitable Wnt polypeptides include wingless (Wg); examples of suitable Wnt receptor polypeptides include Dfz2 (e.g., SEQ ID NO:2).

[0061] The test compound may be effective to inhibit binding between the Wnt polypeptide and the Wnt receptor or to displace the Wnt polypeptide from the Wnt receptor polypeptide. In one embodiment, the Wnt receptor polypeptide is expressed on the surface of a cell (e.g., Drosophila Sneider 2 (S2) cell) transformed with an expression vector encoding said receptor (e.g., Dfz2).

[0062] In another embodiment, the Wnt receptor polypeptide is an N-terminal portion of a full-length Wnt receptor polypeptide, the N-terminal portion including the cysteine-rich amino-terminal domain. In one embodiment, the N-terminal portion is part of a fusion with, e.g., the constant domain of human IgG.

[0063] These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0064]FIG. 1 shows a sequence comparison of Dfz1 and Dfz2.

[0065]FIG. 2 shows hydropathy profiles of mammalian and nematode frizzled homologs.

[0066]FIG. 3 shows a computer-generated image of the expression of DFz2 during Drosophila development evaluated by Northern blot.

[0067]FIG. 4 is a computer-generated image showing that transfection of DFz2 into S2 cells confers a response to Wg protein.

[0068]FIG. 5 is a computer-generated image made using confocal immunomicroscopy showing binding of Wg protein to Dfz-2 transfected cells.

[0069]FIG. 6 is a computer-generated image showing the binding of metabolically labeled Wg protein to a Dfz-2/Ig fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

[0070] I. Definitions

[0071] An “isolated” polypeptide, polynucleotide or antibody refers to such a polypeptide, polynucleotide or antibody that has been at least partially purified away from other cellular material.

[0072] A polynucleotide sequence or fragment is “derived from” another polynucleotide sequence or fragment when it contains the same sequence of nucleotides as are present in the sequence or fragment from which it is derived. For example, a bacterial plasmid contains an insert “derived from” a selected human gene if the sequence of the polynucleotides in the insert is the same as the sequence of the polynucleotides in the selected human gene.

[0073] Similarly, a polypeptide sequence or fragment is “derived from” another polypeptide sequence or fragment when it contains the same sequence of amino acids as are present in the sequence or fragment from which it is derived. A polypeptide “derived from” a nucleic acid is a polypeptide encoded by that nucleic acid. For example, a Wnt receptor polypeptide derived from the human genome (also termed “human Wnt receptor polypeptide” or “hWntR”) is a polypeptide encoded by an mRNA (or corresponding cDNA) transcribed from a human Wnt receptor gene.

[0074] Percent (%) identity, with respect to two amino acid sequences, refers to the % of residues that are identical in the two sequences when the sequences are optimally aligned and no penalty is assigned to “gaps”. In other words, if a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the % identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the “gap” of the first sequence). Optimal alignment is defined as the alignment giving the highest % identity score. Such alignments can be preformed as described herein using the “GENEWORKS” program. Alternatively, alignments may be performed using the local alignment program LALIGN with a ktup of 1, default parameters and the default PAM. The LALIGN program is found in the FASTA version 1.7 suite of sequence comparison programs (Pearson and Lipman, 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, Va.).

[0075] A full-length Wnt receptor (WntR) polypeptide is defined herein as a polypeptide that is a member of the frizzled protein family, encodes a full-length protein, and has at least about a 90% identity with one or more of the following sequences: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0076] II. Overview of the Invention

[0077] The present invention is based on the discovery of a set of novel members of the vertebrate frizzled family of polarity genes, and on the recognition that the frizzled family of polarity genes encodes the receptors for the Wnt family of proteins. The invention is further enhanced by the recognition that the full-length sequence of each member of the frizzled protein family generally shares a substantially greater degree of homology with the full-length sequences of corresponding frizzled proteins in other species (typically about 80% to >95%) than it does with the full-length sequences of other members of the frizzled protein family in the same species (typically about 30% to 60%). Different members of the frizzled family, however, do contain regions within the coding sequences that have high degrees of homology (up to 90% or more) with one another. This feature, combined with similar sizes and hydrophobicity profiles, facilitates the identification of novel members of the frizzled gene family.

[0078] Discoveries described herein enable a number of uses and application of the present invention. These uses and applications are exemplified and discussed in detail below.

[0079] III. Identification of Dfz2 as the Wg Receptor

[0080] Experiments performed in support of the present invention and described in Examples 1-6, below, indicate that Drosophila frizzled gene 2 (Dfz2) is a receptor for wingless (Wg). Example 1 details the cloning of Dfz2, the sequence of which is illustrated in FIG. 1. Hydrophobicity profiles of additional frizzled family members isolated as part of the present invention are shown in FIG. 2. Their sequences are presented in the Sequence Listing. Example 2 describes in situ hybridization experiments to determine the pattern of Dfz2 expression. Example 3 describes Northern analyses (FIG. 3) showing that Dfz2 is expressed throughout development.

[0081] In Example 4, below, Drosophila Sneider 2 (S2) cells were transformed with a Dfz2 expression vector and the effects of the Dfz2 ligand, Wg, were assessed by measuring the levels of armadillo (Arm) protein in response to Wg application (Peifer, et al., 1994; Riggleman, et al., 1990; Van Leeuwen, et al., 1994). The results, shown in FIG. 4, demonstrate that all four Dfz2-transfected S2 cell lines tested showed increased armadillo signal in response to Wg, whereas no such effect was observed with untransfected S2 cells. These results demonstrate that Dfz2 acts as a signal transducing molecule for Wg, consistent with it being a receptor for Wg.

[0082] Further support is provided by immunohistochemical analyses described in Example 5. These experiments were designed to address whether Wg was capable of binding to the Dfz2-transfected cells. Dfz2-transfected and nontransfected cells were exposed to medium containing Wg protein, washed, stained with an anti-Wg antiserum and a labelled secondary antibody, and imaged using a confocal microscope. Exemplary images, shown in FIGS. 5A-5F, demonstrate that approximately 80% of Dfz2-transfected S2 cells exposed to Wg protein stained brightly (FIG. SD) whereas Dfz2-transfected cells in the absence of Wg protein (FIG. 5A) as well as non transfected S2 cells (FIG. 5B) did not. The ability of Wg to bind was also tested in human 293 cells, which are heterologous to the Dfz2 protein. As shown in FIG. 5F, about 10-20% of the transfected cells remained positive, similar to the transfection efficiency of 293 cells. Since 293 cells are of human origin, these results indicate that Wg binds to Dfz2 itself, rather than to a molecule whose expression is induced by Dfz2.

[0083] The binding of Wg protein to Dfz2 was further confirmed using a fusion protein containing the cysteine-rich amino-terminal domain of Dfz2, linked to the constant domain of human IgG, as described in Example 6. The fusion protein or IgG control was added to conditioned medium from normal S2 cells, or S2 cells producing Wg (HS-wg/S2), which had been metabolically-labeled with [³⁵S] cysteine and methionine.

[0084] The fusion proteins and possible complexes were then isolated and analyzed by gel electrophoresis and fluorography (FIG. 6). Two bands of approximately 52 kd (the size of Wg) were detected in the lane with the Dfz2-Ig fusion added to the medium of HS-wg/S2 cells.

[0085] The above results taken together, particularly the observations that (i) Wg binds to DFz2, and (ii) the binding leads to a biological response, strongly support the role of Dfz2 as the receptor for the Wg protein.

[0086] IV. Novel Frizzled Family Members Identified in Vertebrates

[0087] Experiments performed in support of the present invention have further resulted in the identification of at least six novel frizzled family members, termed Wnt receptors (WntRs) herein, in human and mouse. This brings the total number of frizzled-like sequences identified in mammalian genomes to 8, since two (Rfz1 and Rfz2) were previously cloned from rat (Chan, et al., 1992). The six novel genes include Mfz3, Mfz4, Mfz6, Mfz7, and Mfz8, as well as human sequences Hfz3, Hfz5 and Hfz7. A sequence 95% identical over 143 amino acids to Hfz5 was PCR-amplified (Mullis, 1987; Mullis, et al., 1987) from mouse genomic DNA using Hfz5-specific primers, suggesting that an Mfz5 gene exists as well. The DNA and translated amino acid sequences of these 6 family members are provided in the Sequence Listing, along with the sequence of a novel family member isolated from C. elegans (Cfz1). The hydrophobicity profiles of these sequences are presented in FIG. 2. These profiles, along with the sequences of regions that are conserved among different frizzled family members, are used in determining whether a polypeptide sequence is a member of the frizzled gene family. According to the present invention, member of this family are considered to be Wnt receptors.

[0088] Using the guidance herein, one of skill in the art can isolate additional members of the frizzled gene family. In particular, probes homologous to regions conserved among the various family members can be designed and used to probe cDNA or genomic DNA libraries. Hybridization techniques for isolating related sequences are known in the art (e.g., Ausubel, et al., 1989; Sambrook, et al.). These techniques employ “selective hybridization” to detect sequences similar to the probe sequence with high specificity and low background. Selective hybridization is achieved by increasing the stringency of the wash following hybridization, which can in turn be accomplished through the manipulation of salt concentration and/or temperature of the wash buffer (typically 0.1-10×SSC, 0.1% SDS). Lower salt and higher temperatures result in more stringent hybridization conditions.

[0089] Selective hybridization conditions are typically determined empirically for each new probe using several rounds of washes at increasing wash temperatures. The filter or membrane containing the test sequences is evaluated for probe signal after each wash. Wash conditions that give a strong positive signal for a control probe sequence with little or no background signal for unrelated sequences are termed “selective hybridization conditions. Oligonucleotides as small as 16-18 nucleic acids in length can be used to identify similar sequences using hybridization. Of course, the method works well for longer polynucleotides as well.

[0090] Alternatively or in addition, PCR primers corresponding to such conserved regions may be designed and used to isolate additional sequences from any suitable source of DNA, including libraries and reverse transcription (RT)-generated cDNA samples.

[0091] Exemplary WntR polynucleotide sequences are provided herein as sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:15. These or other WntR polynucleotides may be used to recombinantly produce WntR polypeptides using standard expression vectors known in the art. Many such vectors are available commercially (from Invitrogen, Clontech, or Stratagene). Alternatively, WntR polypeptides may be isolated from native sources or produced synthetically.

[0092] V. Wnt Genes and Proteins

[0093] Wg in Drosophila is part of larger gene family (Eisenberg, et al., 1992; Graba, et al., 1995; Russell, et al., 1992) of Wnt genes. At least 3 homologous genes have been identified in Drosophila, and over 10 Wnt genes have been identified in most vertebrates (Nusse and Varmus, 1992). According to the present invention, the products of these genes are the ligands for receptors encoded by the large family of fz-like genes in vertebrates. Determination of which Wnt gene products are specific to which Wnt receptor may be performed by one of skill in the art following the teachings of the present specification.

[0094] All members of the Wnt family encode secreted proteins that act as cell-cell signaling molecules. Wnt genes play an important role in the control of cell growth, particularly in the mammary gland, and can act as oncogenes in mouse mammary tumors. The proteins contain a signal sequence, one or several N-linked glycosylation sites and many cysteine residues. The product of the mouse Wnt-1 gene has been studied most extensively. If Wnt-1 is overexpressed in various cell lines, the protein enters the secretory pathway. The protein can be detected in protease resistant structures, presumably secretory vesicles, and contains carbohydrate structures at several N-linked glycosylation sites. It is thus generally assumed that the Wnt-1 protein is secreted from cells, although extracellular forms of the protein have been difficult to detect. In addition, most of the intracellular Wnt-1 protein made in transfected cells is incompletely glycosylated (it remains sensitive to endoglycosidase H) and has probably not traversed the Golgi apparatus. Moreover, much of the Wnt-1 protein becomes associated with the resident ER protein BiP, indicating that it is incorrectly folded.

[0095] In spite of these difficulties, it has been shown that Wnt-1 overproduction leads to secretion of modest amounts of extracellular protein. The secreted forms have undergone more extensive glycosylations, and may bind to the cell surface or to the extracellular matrix.

[0096] VI. Role of Wnt in Cancer

[0097] Members of the Wnt gene family are important regulators of mammary cell growth. Indeed, Wnt genes owe their discovery to their role as oncogenes in mouse mammary cancer: previous experiments which examined the sequence around integration sites for Mouse Mammary Tumor Virus (MMTV) DNA showed that many tumors had sustained proviral insertions near the Wnt-1 gene, the first member of this gene family. A biological assay for Wnt-1 was subsequently established using gene transfer experiments. This assay was used to show that certain mammary gland-derived cell lines can be morphologically transformed by Wnt-1. Direct evidence that Wnt-1 expression gives a strong growth stimulus to mammary cells came from transgenic mice carrying Wnt-1 linked to the MMTV promoter, which developed mammary hyperplasia and tumors. By infecting primary mammary cells with retroviruses expressing Wnt-1 and re-implantation of the infected cells, similar hyperplasia of the mammary gland were obtained. Additional experiments led to the identification of a Wnt-1 related oncogene activated by MMTV insertion, called Wnt-3.

[0098] The growth stimulus generated by the expression of Wnt-1 in the mammary gland implies that mammary cells are equipped with a Wnt receptor that becomes activated by the Wnt-1 protein, as well as the other signaling components. While neither Wnt-1 nor Wnt-3 are expressed in the normal mammary gland, at least 5 other Wnt genes are expressed during specific stages of mammary gland development, including during the rapid expansion of the pre-lactating gland or when the gland regresses.

[0099] The oncogenic action of Wnt-1 and Wnt-3 is best explained by their acting as ligands for Wnt receptors meant for other Wnt genes, and activating these receptors inappropriately. Alternatively, Wnt-1 and Wnt-3 may not activate these receptors but may interfere with a ligand-receptor interaction normally leading to regression of the gland.

[0100] The strong growth stimulus by oncogenic Wnt genes and the dynamic expression patterns of other Wnt genes in the mammary gland provide evidence that Wnt genes are important regulators of mammary gland growth. In analogy with the mouse, it is likely that some Wnt genes expressed during the normal cycles of growth of the mammary gland. In contrast to silent genes, genes that are expressed are candidates to become amplified, since the ensuing overexpression of those genes can give a selective advantage to cells even during the first rounds of amplification.

[0101] A survey of mouse mammary tumors identified one tumor where the mouse Wnt-2 gene was amplified and overexpressed whereas Wnt-2 had a low level of expression in the normal gland. Further, there was no evidence for insertion of MMTV near Wnt-2 in that tumor. This finding shows that Wnt genes are not necessarily activated only by MMTV, a relevant factor for human breast cancer since that disease has no viral etiology but is often characterized by gene amplification. In view of these observation, it is contemplated that inhibitors of Wnt/WntR interactions may be used to inhibit the growth and/or spread of breast and other types of cancer.

[0102] VII. Screening Methods

[0103] In view of the role of Wnt in cancer and other processes involving growth, development and proliferation (both normal and abnormal), it would be desirable to identify modulators of Wnt activity that affect the interactions of specific Wnt proteins with their receptors. Such modulators may, for example, inhibit the binding of Wnt to its receptor (e.g., by competitive or noncompetitive inhibition), or they may potentiate or stabilize the binding. The recognition that members of the frizzled family of proteins can act as receptors for the Wnt family of proteins enables a number of screening approaches to the isolation of such modulatory compounds that have heretofore not been possible.

[0104] Examples of such screening approaches include protein-protein binding assays in which the level of binding of Wnt to its receptor, or a biological consequence of such binding, is measured. The latter assay is exemplified in Example 4, where cells not normally expressing Wnt receptors are transformed with a Wnt receptor (in this case, Dfz2), and the effects of Wnt (in this case, Wg) on the cells are measured (in this case, by detecting levels of Arm). Such cells may be transformed with the Wnt receptor of choice (e.g., any of fz1, fz2, fz3, fz4, fz5, fz6, fz7 or fz8 receptors).

[0105] In Example 4, expression of Arm was detected using a Western blot method. Other methods may be employed which are more suitable for high throughput screening applications. For example, labelled anti-Arm antibodies may be used to directly visualize levels of Arm in multi-well format screen.

[0106] Alternatively, the assays may simply detect the degree of binding between Wnt ligands and Wnt receptors, and not the biological consequences of such binding. For example, cells expressing a selected Wnt receptor may be plated in the wells of a 96-well plate and contacted with a solution containing reporter-labeled Wnt (e.g., radiolabelled of fluorescently-tagged) in the presence and absence of a test compound (i.e., a putative modulator of Wnt/receptor interactions). The effect of the test compound on the extent of binding between Wnt and Wnt receptor is measured, and the compound is identified as effective if its effect on the extent of binding is above a threshold level (e.g., a several-fold difference in binding level between control and experimental samples) In one embodiment, the threshold is a 2-fold difference. In another embodiment, it is a 5-fold difference. In yet another it is a 10-fold or greater difference. The difference in binding in the presence and absence of an effective test compound is preferably statistically-significant, as determined by a standard statistical test.

[0107] It will be appreciated that the putative modulator compound can alternatively be added after the cells had been incubated with labelled Wnt. In a screen for inhibitors of binding, the system is assayed for a decrease in the signal reflecting bound labelled Wnt, or an increase in the signal reflecting labelled Wnt in solution.

[0108] Such a screen may also be employed to screen for potentiators of Wnt/receptor interactions. For example, test compounds may be added to the wells (either during or after incubation with labelled Wnt), and the wells-then contacted with unlabeled Wnt. Test compounds in wells where the unlabelled Wnt is less effective at displacing the bound labelled Wnt are selected for more detailed examination of ability to potentiate Wnt/receptor binding.

[0109] Assays such as described above may also be used to determine the relationship between different Wnt proteins and different receptors. For example, the ligand concentration dependence of binding may be used in measurement of the relative affinities of selected Wnt receptors with selected ligands, and ligands with a selected affinity for the receptor can be examined further using, e.g., in vitro or in vivo assays. In this manner, one of skill in the art can identify which Wnt protein(s) is optimally paired with which receptor(s).

[0110] In cases where the Wnt ligand has been matched to a specific Wnt receptor (e.g., in the case of Wg and Dfz2), the receptor/ligand pair can be used in, e.g., screening applications. For example, the pair may be used in a binding assay to screen for compounds which are effective to modulate the binding of the specific ligand to its receptor. These methods enable the identification of compounds with two general types of activities: (i) those which act generally, e.g., on a class of Wnt/Wnt receptor pairs, to disrupt or facilitate binding, and (ii) those which act selectively disrupt or facilitate the binding between a selected Wnt ligand and its receptor, but not between other Wnt ligands and their receptors.

[0111] Compounds identified by one of the screens described herein may be further evaluated for efficacy using an in vitro assay such as described above. Further, such compounds may be tested in in vivo models employing Wnt/Wnt receptor interactions. For example, the compounds may be tested in a mouse mammary tumor model for effectiveness at inhibiting growth of mammary tumors.

[0112] VIII. Compounds Suitable for Screening

[0113] A variety of different compounds may be screened using methods of the present invention. They include peptides, macromolecules, small molecules, chemical and/or biological mixtures, and fungal, bacterial, or algal extracts. Such compounds, or molecules, may be either biological, synthetic organic, or even inorganic compounds, and may be obtained from a number of sources, including pharmaceutical companies and specialty suppliers of libraries (e.g., combinatorial libraries) of compounds.

[0114] In cases where an identified active compound is a peptide, the peptide may be utilized to design a peptoid mimetic and aid in the discovery of orally-active small molecule mimetics. Alternatively, the peptides themselves may be used as therapeutics.

[0115] Further, the structure of a bioactive polypeptide may be determined using, for example, NMR, and may be used to select the types of small molecules screened.

[0116] Methods of the present invention are well suited for screening libraries of compounds in multi-well plates (e.g., 96-well plates), with a different test compound in each well. In particular, the methods may be employed with combinatorial libraries. A variety of combinatorial libraries of random-sequence oligonucleotides, polypeptides, or synthetic oligomers have been proposed (Kramer, et al., 1993; Houghten, 1985, 1994; Houghten, et al., 1986, 1991, 1992; Ohlmayer, et al., 1993; Dooley, et al., 1993a-1993b; Eichler, et al., 1993; Pinilla, et al., 1992, 1993; Ecker, et al., 1993; and Barbas, et al., 1992). A number of small-molecule libraries have also been developed (e.g., Bunin, et al., 1994; Bunin and Ellman, 1992; Virgilio and Ellman, 1994).

[0117] Combinatorial libraries of oligomers may be formed by a variety of solution-phase or solid-phase methods in which mixtures of different subunits are added stepwise to growing oligomers or parent compound, until a desired oligomer size is reached (typically hexapeptide or heptapeptide). A library of increasing complexity can be formed in this manner, for example, by pooling multiple choices of reagents with each additional subunit step (Houghten, et al., 1991).

[0118] Alternatively, the library may be formed by solid-phase synthetic methods in which beads containing different-sequence oligomers that form the library are alternately mixed and separated, with one of a selected number of subunits being added to each group of separated beads at each step (Furka, et al., 1991; Lam, et al., 1991, 1993; Zuckermann, et al., 1992; Sebestyen, et al., 1993).

[0119] The identity of library compounds with desired effects on the binding of a Wnt to a Wnt receptor can be determined by conventional means, such as iterative synthesis methods in which sublibraries containing known residues in one subunit position only are identified as containing active compounds.

[0120] IX. Pharmaceutical Preparations of Active Compounds

[0121] After identifying certain test compounds as potential WntR agonists or antagonists agents, the practitioner of the screening assay will typically continue to test the efficacy and specificity of the selected compounds both in vitro and in vivo. Whether for subsequent in vivo testing, or for administration to an animal as an approved drug, agents identified in the screening assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.

[0122] The compounds selected in the screening assay, or a pharmaceutically acceptable salt thereof, may accordingly be formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glyccrol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, “biologically acceptable medium” includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the compound, its use in the pharmaceutical preparation of the invention is contemplated.

[0123] Suitable vehicles and their formulation inclusive of other proteins are described, for example, in Gennaro, 1990. These vehicles include injectable “deposit formulations”. Based on the above, such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the compound in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids. In a preferred embodiment, the compound can be disposed in a sterile preparation for topical and/or systemic administration. In the case of freeze-dried preparations, supporting excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH. Similar solutions may also be used for the pharmaceutical compositions in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH (for example, neutral pH).

[0124] The following examples illustrate but in no way are intended to limit the present invention.

Materials and Methods

[0125] Unless otherwise indicated, restriction enzymes and DNA modifying enzymes were obtained from New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.). Nitrocellulose paper was obtained from Schleicher and Schuell (Keene, N.H.). Other chemicals were purchased from Sigma (St. Louis, Mo.) or United States Biochemical (Cleveland, Ohio). Unless otherwise specified, the experiments were performed using standard methods (Ausubel, et al., 1988; Sambrook, et al., 1989; Harlow, et al., 1988).

[0126] A. Buffers

[0127] Phosphate-buffered saline (PBS)

[0128] 10×stock solution, 1 liter:

[0129] 80 g NaCl

[0130] 2 g KCl

[0131] 11.5 g Na₂HPO4-7H₂O

[0132] 2 g KH₂PO₄

[0133] Working solution, pH 7.3:

[0134] 137 mM NaCl

[0135] 2.7 mM KCl

[0136] 4.3 mM Na₂HPO₄-7H₂O

[0137] 1.4 mM KH₂PO₄

EXAMPLE 1 Molecular Cloning of DFz2

[0138] Polymerase chain reaction (PCR; Mullis, 1987; Mullis, et al., 1987) primer pools YW157 and YW158 were designed based on sequences (SEQ ID NO:16, SEQ ID NO:17, respectively) conserved in Dfz1, Human frizzled 3 (Hfz3), Rat frizzled 1 (Rfz1) and Rat frizzled 2 (Rfz2). The primer pools were completely degenerate, that is, each possible codon of each amino acid in SEQ ID NO:16 and SEQ ID NO:17 was represented in the respective primer pool, with the exception that the wobble base of the 3′-most codon was not included in YW157. The primers were used to amplify Drosophila genomic DNA, resulting in an amplification product that, when sequenced, was found to contain a novel frizzled family member—Dfz2. The PCR product was used to isolate genomic clones of Dfz2 from an adult Drosophila genomic library (Maniatis, et al.) and cDNA clones from a 0-24 hr cDNA library.

[0139] The amino acid sequence of Dfz2 was compared to that of Dfz1 by aligning the sequences as shown in FIG. 1. Dfz2 and Dfz1 are 32% identical. Identical residues are indicated in the consensus and the conserved cysteine residues in the cysteine-rich domain are in bold-face. The sequence alignments were done using the “GENEWORKS” program.

[0140] Hydropathy values were calculated using the “MACVECTOR” 3.5 software according to the Kyte-Doolittle software and a window size of 15 amino acids.

EXAMPLE 2 In Situ RNA Hybridization

[0141] In situ hybridization experiments were performed to determine the pattern of Dfz2 expression. Freshly dissected adult brains, whole embryos or heads were rapidly frozen in plastic molds placed on a dry ice/alcohol slurry and processed for sectioning as described previously (Cole, et al., 1990). ³⁵S-Labeled antisense riboprobes were prepared from linearized p“BLUESCRIPT” plasmid subclones using either T3 or T7 RNA polymerase. In situ hybridization was performed as described by Saffen, et al., and hybridized sections were exposed to X-ray film and digitized.

EXAMPLE 3 Expression of DFz2 During Drosophila Development

[0142] The expression pattern of DFz2 was assessed using Northern (RNA) blot analysis. Total RNA was isolated using the LiCl-Urea precipitation method (Auffray and Rougeon, 1980). 30 microgram of RNA from each sample was resolved on a formaldehyde 1% agarose gel. The RNA was transferred to a nylon filter, cross-linked by UV irradiation and hybridized to a probe made by random priming Dfz2 or RP49 DNA fragments using standard methods (Sambrook, et al., 1989). In other experiments, Poly (A)⁺ RNA from various stages of Drosophila development was first selected from total RNA using the Invitrogen “FASTTRACK” 2.0 kit and 5 μg was loaded per lane.

[0143] Exemplary results are shown in FIG. 3. A 4.0 kb transcript was detected in embryonic stages 0-2; 2-3; 4-5; 9-12, first, second and third instar larvae and pupae. A transcript of similar size was observed in Drosophila clone-8 cells (cl-8), a cell line from imaginal discs previously shown to be responsive to Wg activity in vitro. Drosophila Schneider 2 (S2) cells, which do not respond to Wg, did not contain detectable DFz2 transcripts. The blot was also probed for expression of the ribosomal protein RP49 (O'Connell and Rosbash, 1994, lower panel) as a control for RNA integrity and loading.

EXAMPLE 4 Transfection of DFz2 in S2 Cells Confers a Response to Wg Protein

[0144] S2 cells were evaluated for Dfz2 expression because the cells are known not to respond to Wg (Yanagawa, et al., 1995). Since, as described above, the native cells did not express Dfz2, they were used in Dfz2 transfection experiments to determine whether expression of Dfz2 would confer sensitivity to Wg.

[0145] An expression vector containing DFz2 coding sequences under the control of a metal-inducible metallothionein promoter was used to transfect S2 cells using standard methods. Stable cell lines were derived by selection in hygromycin and tested for Dfz2 expression. In cells grown in the absence of inducers, a baseline level of expression was detected with an antiserum to Dfz2. Induction of the metallothionein promoter resulted in increased levels of expression.

[0146] Sensitivity of the Dfz2-transfected S2 cells to Wg protein was assessed by measuring the levels of armadillo (Arm) protein in response to Wg application. In intact Drosophila embryos and in clone-8 cells, Arm protein migrates in two different forms, differing from each other in phosphorylation. When these cells are incubated in the presence of soluble Wg protein, the level of the faster migrating (non-phosphorylated) form increases (Peifer, et al., 1994; Riggleman, et al., 1990; Van Leeuwen, et al., 1994). This increase can be detected using a standard Western blot assay as described below.

[0147] Conditioned medium containing Wg protein was produced by subjecting S2HSwg cells to heat-shock for 30 minutes at 37° C., allowing the cells to recover for 30 minutes at 25° C., and resuspending them in S2 medium without fetal calf serum (FCS). The cells were incubated for 3 hrs to allow secretion of proteins into the medium, after which they were removed by centrifugation (10 min., 2000×g and 1hr, 100,000×g, respectively). The conditioned media were concentrated 12-fold (“CENTRIPREP30”, Amicon) and used in the experiments as follows.

[0148] Clone 8, untransformed S2, and Dfz-transformed S2 (S2Dfz2) cells were incubated for 2 hrs in 6-well dishes in either normal concentrated medium or in concentrated medium from S2 cells producing Wg. Overexpression of the Dfz2 gene (under control of the metallothionein promoter) was induced by culturing S2Dfz2 and S2 control cells in S2 medium containing 0.5 mM CuSO₄ for 5 hrs prior to the incubation with the conditioned media.

[0149] The target cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40, 5 mM EDTA) supplemented with 20 μg leupeptin, 100 μg aprotinin and 180 μg PMSF per ml. The extracts were subjected to electrophoresis and Western blotting. Blots were stained in Ponceau Red to evaluate equal loading of total protein and transfer, and then incubated overnight in blocking buffer with monoclonal anti-arm antibody 7A1 at a 1:1000 dilution or rat-polyclonal anti-α-catenin antibody DCAT-1 (Oda, et al., 1993), diluted 1:1000. The blots were washed three times for 15 min each in TBST and incubated for 1 hr with horseradish peroxidase conjugated secondary antibodies (Biorad) diluted 1:20,000 in blocking buffer.

[0150] Incubation of DFz2-transfected S2 cells (but not untransfected S2 cells) in the presence of soluble Wg protein resulted in an increase in the level of Arm protein similar to that observed in Drosophila embryos and clone-8 cells. Exemplary results are shown in FIG. 4. Addition of Wg (wingless) results in increased signal intensity of the armadillo band. No such effect is observed with untransfected S2 cells. However, all four independent Dfz2-transfected S2 cell lines, derived from two separate transfections, showed increased armadillo signal in response to Wg (two of the four are shown). Further induction of Dfz2 expression by copper sulphate in the transfected cells led to a slight decrease in the response to Wg. As a control for equal loading, the blots were stripped and incubated with an antiserum against α-catenin (lower panel).

EXAMPLE 5 Wg Protein Binds to Dfz2 Transfected Cells

[0151] The results described in Example 4 showed that Dfz2 acts as a signal transducing molecule for Wg, suggesting that it is a receptor for Wg. Immunohistochemical analyses were performed to determine whether Wg was capable of binding to the Dfz2-transfected cells.

[0152] Nontransfected Sneider 2 (S2) cells and S2 cells expressing Dfz2 were washed twice in PBS and incubated with 1.5 ml of medium alone or 1.5 ml of a 10×concentrated stock of Wg conditioned medium at 4° C. for 3 hours. After three 10 minute washes with PBS, the cells were fixed in 2% methanol-free formaldehyde (Polysciences, Inc) for 15 minutes at room temperature. Following three more 10 minute washes with PBS, affinity purified Wg antibody at {fraction (1/25)} and 5 donkey serum were added to the cells in PBS and incubated overnight at 4° C.

[0153] The antiserum was affinity-purified using a bacterial fusion protein containing a domain unique to Wg (the Wg insert—an 85 amino acid sequence not found in any wg orthologs). Previous experiments have indicated that this domain is dispensable for Wg activity, that it probably does not participate in the interactions between Wg and its receptor.

[0154] Following 3 additional 10 minute washes, fluorescent-labeled cy3 secondary antibody, donkey anti-rabbit (Sigma), at {fraction (1/100)} and 5 donkey serum were added to the cells for 1 hour at room temperature. The cells were then washed 3 more times in PBS and mounted in Vectashield mounting medium (Vector).

[0155] Confocal images were collected with a Bio-Rad MRC 1000 confocal laser attached to a Zeiss Axio scope microscope. Exemplary images are shown in FIGS. 5A-5F. Normal and transfected cells were incubated with either normal S2 medium (FIG. 5A) or concentrated conditioned medium from S2 cells producing Wg (FIGS. 5B, 5C, 5D, 5E, 5F). Dfz2-transfected S2 cells stained brightly in approximately 80% of the cells when incubated with Wg and the antiserum (FIG. 5D) whereas Dfz2-transfected cells in the absence of Wg protein (FIG. 5A) as well as non transfected S2 cells (FIG. 5B) showed only some spots of background staining. The positive staining was not uniform over the cell surface but punctate and may reflect clustering of receptor complexes.

[0156] The ability of Wg to bind was also tested in heterologous cells (human 293 cells) transiently-transfected with Dfz2. In view of high background binding observed in initial experiments, the transiently-transfected 293 cells were preincubated with chlorate, which inhibits sulfation of proteins and glucosaminoglycans, and with heparatinase, to remove heparin-like molecules. This pre-treatment significantly lowered the background binding (presumably due to Wg binding to extracellular matrix; FIG. 5E). As shown in FIG. 5F, about 10-20% of the transfected cells remained positive, similar to the transfection efficiency of 293 cells. Since 293 cells are of human origin, these results strongly suggest that Wg binds to Dfz2 itself, rather than to a molecule whose expression is induced by Dfz2.

[0157] In contrast to the positive staining patterns observed with Dfz2-transfected cells, no staining was detected in S2 cells expressing Notch (FIG. 5C). Notch is a protein that has been previously proposed to act as a receptor for Wg (Couso and Arias, 1994).

[0158] The above results taken together indicate that Wg protein can specifically bind to cells expressing Dfz2, and that this binding is not likely due to clonal variation.

EXAMPLE 6 Binding of Metabolically-Labeled Wg Protein to a Dfz-2/IgG Fusion Protein

[0159] The binding of Wg protein to Dfz2 itself was also assayed using a fusion protein containing the cysteine-rich amino-terminal domain of Dfz2, linked to the constant domain of human IgG. The fusion protein or IgG control was added to conditioned medium from normal S2 cells, or S2 cells producing Wg (HS-wg/S2), which had been metabolically-labeled with [³⁵S] cysteine and methionine.

[0160] The fusion proteins and possible complexes were then retrieved by adding sepharose-ProteinA beads and analyzed by gel electrophoresis and fluorography. FIG. 6 shows that the Dfz2 fusion protein, but not the control IgG, selectively binds to labeled proteins of 52 kD, the size of the mature Wg protein. Normal S2 cells did not produce Dfz-2 binding proteins.

[0161] While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.

1 18 2344 base pairs nucleic acid double linear DNA (genomic) NO NO Dfz2 Polynucleotide, coding region begins at nucleotide #225 1 GCGCTGTGTC TGAAGGAAAC ACTACCCGCT TTTCCGGCTC TCGAGGCGCC TCCACGAAGG 60 AGTGAGGTGC AACCGCAGAG AAGGTCAGCA AAGAAAGAGC AAGGGGTTCC AAGTCACACA 120 ACCGAACTAA GCTAAGACGC ACAAAATGAG ACACAATCGA CTGAAGGTCC TGATCCTGGG 180 ACTCGTCCTC CTGCTGACAT CTTGTCGAGC GGATGGACCG CTGCACAGTG CGGATCACGG 240 CATGGGCGGA ATGGGCATGG GTGGTCACGG CCTGGACGCG AGTCCCGCAC CCGGTTACGG 300 AGTGCCAGCC ATACCCAAGG ATCCCAATCT GCGATGCGAG GAGATCACCA TACCAATGTG 360 TCGGGGCATT GGCTACAACA TGACATCCTT CCCCAACGAA ATGAACCATG AGACCCAGGA 420 CGAAGCGGGC CTGGAGGTGC ACCAGTTCTG GCCCCTGGTG GAGATCAAAT GCTCGCCGGA 480 CCTCAAGTTC TTCCTGTGCA GCATGTACAC GCCCATCTGC CTGGAGGATT ACCACAAGCC 540 GCTGCCCGTT TGCCGGAGTG TCTGCGAGAG AGCCCGCTCG GGATGCGCAC CCATCATGCA 600 GCAGTACAGC TTCGAATGGC CGGAGAGAAT GGCGTGCGAG CACTTGCCCC TTCATGGTGA 660 CCCCGACAAT CTGTGCATGG AACAGCCCTC GTACACGGAG GCTGGCAGCG GTGGCAGCTC 720 GGGCGGATCG GGTGGCTCTG GCAGCGGTTC CGGCTCCGGC GGCAAACGGA AGCAAGGAGG 780 CAGTGGCTCG GGCGGCAGTG GGGCCGGCGG CAGCAGCGGT TCCACCTCAA CGAAGCCGTG 840 CCGCGGACGC AATTCAAAAA ACTGCCAAAA TCCCCAAGGA GAAAAGGCAA GCGGAAAAGA 900 GTGCAGCTGC TCGTGCCGCT CCCCACTCAT CTTCCTGGGG AAGGAGCACT GGCTGCAGCA 960 GCAGTCGCAG ATGCCCATGA TGCACCATCC ACACCACTGG TACATGAACC TCACTGTCCA 1020 AAGGATCGCC GGCGTTCCAA ACTGCGGCAT ACCGTGCAAG GGGCCCTTCT TCAGCAACGA 1080 CGAAAAGGAT TTCGCCGGCC TCTGGATCGC CCTGTGGTCG GGACTGTGCT TCTGCAGCAC 1140 GCTCATGACC CTAACCACAT TCATCATCGA CACCGAAAGG TTTAAGTACC CGGAGCGGCC 1200 ATTGTCTTCC TCTCCGCCTG CTACTTCATG GTGGCAGTGG GCTACCTGTC GCGCAACTTC 1260 CTGCAGAACG AGGAGATCGC CTGCGACGGC CTGCTGCTCC GGGAAAGCTC CACGGGTCCG 1320 CACTCTTGCA CCCTGGTCTT CCTGCTCACC TACTTCTTTG GCATGGCCTC GTCCATCTGG 1380 TGGGTGATCC TCACTTTCAC CTGGTTCCTG GCCGCTGGTC TGAAGTGGGG CAATGAGGCC 1440 ATCACCAAGC ACTCGCAGTA CTTCCATCTG GCCGCCTGGT TGATTCCCAC TGTCCAGTCC 1500 GTGGCCGTAC TCCTGCTCTC GGCGGTGGAT GGCGATCCCA TTCTGGGCAT CTGCTATGTG 1560 GGCAACCTCA ATCCGGATCA CCTAAAGACC TTTGTGCTGG CCCCGCTCTT AGTTTACCTC 1620 GTAATCGGCA CCACCTTCCT GATGGCCGGC TTTGTGTCCC TCTTCCGCAT CCGCTCGGTT 1680 ATCAAGCAAC AGGGCGGTGT AGGAGCTGGT GTCAAGGCGG ACAAGCTGGA GAAACTGATG 1740 ATCAGGATTG GCATCTTCTC GGTGCTCTAC ACGGTGCCGG CCACCATAGT TATCGGATGT 1800 TACCTGTACG AAGCAGCCTA CTTTGAGGAC TGGATCAAGG CCCTGGCCTG TCCATGCGCC 1860 CAGGTGAAGG GTCCCGGCAA GAAGCCTCTC TACTCGGTCC TGATGCTCAA GTACTTCATG 1920 GCCCTGGCCG TGGGCATCAC CTCGGGCGTG TGGATCTGGT CTGGCAAGAC GCTGGAGAGC 1980 TGGCGACGCT TCTGGCGGAG ACTCCTAGGA GCGCCGGACC GCACGGGCGC CAACCAGCTG 2040 GCGATCAAGC AGCGGCCTCC GATCCCGCAT CCCTATGCCG GATCTGGAAT GGGCATGCCC 2100 GTGGGCTCGG CGGCGGGCTC CCTGCTGGCC ACGCCCTACA CCCAGGCGGG CGGACGTCGG 2160 TGGCCTCCAC CAGCCACCAC CACCTGCACC ACCACGTTCT CAAGCAGCCG GCGGCCAGCC 2220 ACGTATGACA TGGAGAGTCG GGGGGAGCAT CGACCATGGG CGGCGGTGGG GGCGGCGGTA 2280 CAGCCCTTGG CGGCGGCACC CTGGGCCACG GCACCGCGAT GAGCAGCAGC ACGGTCGGCA 2340 TGGG 2344 694 amino acids amino acid single linear protein NO NO Dfz2 Polypeptide 2 Met Arg His Asn Arg Leu Lys Val Leu Ile Leu Gly Leu Val Leu Leu 1 5 10 15 Leu Thr Ser Cys Arg Ala Asp Gly Pro Leu His Ser Ala Asp His Gly 20 25 30 Met Gly Gly Met Gly Met Gly Gly His Gly Leu Asp Ala Ser Pro Ala 35 40 45 Pro Gly Tyr Gly Val Pro Ala Ile Pro Lys Asp Pro Asn Leu Arg Cys 50 55 60 Glu Glu Ile Thr Ile Pro Met Cys Arg Gly Ile Gly Tyr Asn Met Thr 65 70 75 80 Ser Phe Pro Asn Glu Met Asn His Glu Thr Gln Asp Glu Ala Gly Leu 85 90 95 Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Lys Cys Ser Pro Asp 100 105 110 Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys Leu Glu Asp 115 120 125 Tyr His Lys Pro Leu Pro Val Cys Arg Ser Val Cys Glu Arg Ala Arg 130 135 140 Ser Gly Cys Ala Pro Ile Met Gln Gln Tyr Ser Phe Glu Trp Pro Glu 145 150 155 160 Arg Met Ala Cys Glu His Leu Pro Leu His Gly Asp Pro Asp Asn Leu 165 170 175 Cys Met Glu Gln Pro Ser Tyr Thr Glu Ala Gly Ser Gly Gly Ser Ser 180 185 190 Gly Gly Ser Gly Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Lys Arg 195 200 205 Lys Gln Gly Gly Ser Gly Ser Gly Gly Ser Gly Ala Gly Gly Ser Ser 210 215 220 Gly Ser Thr Ser Thr Lys Pro Cys Arg Gly Arg Asn Ser Lys Asn Cys 225 230 235 240 Gln Asn Pro Gln Gly Glu Lys Ala Ser Gly Lys Glu Cys Ser Cys Ser 245 250 255 Cys Arg Ser Pro Leu Ile Phe Leu Gly Lys Glu Gln Leu Leu Gln Gln 260 265 270 Gln Ser Gln Met Pro Met Met His His Pro His His Trp Tyr Met Asn 275 280 285 Leu Thr Val Gln Arg Ile Ala Gly Val Pro Asn Cys Gly Ile Pro Cys 290 295 300 Lys Gly Pro Phe Phe Ser Asn Asp Glu Lys Asp Phe Ala Gly Leu Trp 305 310 315 320 Ile Ala Leu Trp Ser Gly Leu Cys Phe Cys Ser Thr Leu Met Thr Leu 325 330 335 Thr Thr Phe Ile Ile Asp Thr Glu Arg Phe Lys Xaa Pro Gly Ala Ala 340 345 350 Ile Val Phe Leu Ser Ala Cys Tyr Phe Met Val Ala Val Gly Tyr Leu 355 360 365 Ser Arg Asn Phe Leu Gln Asn Glu Glu Ile Ala Cys Asp Gly Leu Leu 370 375 380 Leu Arg Glu Ser Ser Thr Gly Pro His Ser Cys Thr Leu Val Phe Leu 385 390 395 400 Leu Thr Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp Val Ile Leu 405 410 415 Thr Phe Thr Trp Phe Leu Ala Ala Gly Leu Lys Trp Gly Asn Glu Ala 420 425 430 Ile Thr Lys His Ser Gln Tyr Phe His Leu Ala Ala Trp Leu Ile Pro 435 440 445 Thr Val Gln Ser Val Ala Val Leu Leu Leu Ser Ala Val Asp Gly Asp 450 455 460 Pro Ile Leu Gly Ile Cys Tyr Val Gly Asn Leu Asn Pro Asp His Leu 465 470 475 480 Lys Thr Phe Val Leu Ala Pro Leu Phe Val Tyr Leu Val Ile Gly Thr 485 490 495 Thr Phe Leu Met Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser Val 500 505 510 Ile Lys Gln Gln Gly Gly Val Gly Ala Gly Val Lys Ala Asp Lys Leu 515 520 525 Glu Lys Leu Met Ile Arg Ile Gly Ile Phe Ser Val Leu Tyr Thr Val 530 535 540 Pro Ala Thr Ile Val Ile Gly Cys Tyr Leu Tyr Glu Ala Ala Tyr Phe 545 550 555 560 Glu Asp Trp Ile Lys Ala Leu Ala Cys Pro Cys Ala Gln Val Lys Gly 565 570 575 Pro Gly Lys Lys Pro Leu Tyr Ser Val Leu Met Leu Lys Tyr Phe Met 580 585 590 Ala Leu Ala Val Gly Ile Thr Ser Gly Val Trp Ile Trp Ser Gly Lys 595 600 605 Thr Leu Glu Ser Trp Arg Arg Phe Trp Arg Arg Leu Leu Gly Ala Pro 610 615 620 Asp Arg Thr Gly Ala Asn Gln Ala Leu Ile Lys Gln Arg Pro Pro Ile 625 630 635 640 Pro His Pro Tyr Ala Gly Ser Gly Met Gly Met Pro Val Gly Ser Ala 645 650 655 Ala Gly Ser Leu Leu Ala Thr Pro Tyr Thr Gln Ala Gly Gly Ala Ser 660 665 670 Val Ala Ser Thr Ser His His His Leu His His His Val Leu Lys Gln 675 680 685 Pro Ala Ala Ser His Val 690 2624 base pairs nucleic acid double linear mRNA NO NO Mus musculus frizzled-3 protein, Coding Region 313..2313 3 GAATTCGGCA CGAGAAGATG GAATCTGTGA TTTGGGAATG CGGTTGATGG AGTTGCTATG 60 CTGGCCAGAT GTGCCCAATG TAATAAAATG AAAAGAAGAT ACAAGATGAT GTCATCTTCC 120 CATATTGTGA AACCAAAAAC AAATGCCCTT TGTGAGACCA GGTTACCAGT TCTTTGACAG 180 TACAGGGAGT TTTTAAACTG AGGAGCCTAA CAGATAAGGG GTACTTTCAA GCTGAGACCT 240 GCAGGCATAT ACTGATCTAA AACGCATCTT GTGTAGATCT GATCATCCGA GCCTCATTCT 300 GATCCAGGAA GAATGGCTGT GAGCTGGATT GTCTTTGATC TTTGGCTCTT GACTGTGTTT 360 CTGGGGCAGA TAGGTGGGCA CAGTTTGTTT TCTTGTGAAC CTATAACCTT GAGGATGTGC 420 CAAGATTTGC CTTACAATAC TACCTTCATG CCTAATCTTC TGAACCATTA TGACCAACAG 480 ACTGCAGCTT TAGCAATGGA GCCCTTCCAC CCTATGGTGA ACCTGGATTG TTCTCGGGAT 540 TTTCGGCCAT TTCTTTGTGC ACTCTATGCC CCTATTTGTA TGGAATATGG ACGTGTCACA 600 CTTCCCTGCC GTAGGCTGTG TCAGCGTGCC TATAGCGAGT GTTCAAAACT CATGGAGATG 660 TTTGGTGTCC CGTGGCCTGA AGATATGGAG TGCAGTAGGT TTCCAGATTG TGATGAGCCA 720 TATCCCCGAC TTGTGGATTT GAATTTAGTT GGAGATCCAA CTGAAGGAGC CCCAGTTGCA 780 GTGCAGAGGG ACTATGGTTT TTGGTGTCCC AGAGAGTTAA AAATTGATCC TGATCTTGGC 840 TATTCCTTTC TGCACGTGCG AGATTGTTCG CCACCATGTC CCAATATGTA CTTCAGGAGA 900 GAAGAACTGT CATTTGCTCG CTATTTCATA GGCCTGATTT CAATCATTTG CCTCTCTGCC 960 ACATTGTTTA CTTTTTTAAC CTTTCTAATT GACGTCACAA GATTCCGTTA CCCTGAAAGA 1020 CCTATCATAT TTTATGCAGT CTGCTACATG ATGGTGTCAT TAATTTTCTT CATTGGGTTT 1080 TTGCTGGAGG ACCGAGTAGC CTGCAATGCA TCTAGCCCTG CACAGTATAA GGCTTCTACA 1140 GTGACACAAG GATCTCACAA TAAGGCCTGT ACCATGCTCT TTATGGTACT ATATTTTTTC 1200 ACTATGGCTG GCAGTGTATG GTGGGTAATT CTTACCATCA CATGGTTTTT AGCAGCTGTG 1260 CCAAAGTGGG GCAGTGAAGC TATTGAGAAG AAAGCATTGC TGTTTCATGC CAGTGCCTGG 1320 GGCATCCCCG GAACTCTAAC TATCATCCTT TTAGCGATGA ATAAAATTGA AGGTGACAAT 1380 ATTAGTGGCG TGTGTTTTGT CGGCCTCTAC GACGTTGATG CATTAAGATA TTTCGTTCTC 1440 GCTCCCCTCT GCCTGTATGT GGTAGTTGGG GTTTCTCTCC TTTTAGCCGG CATTATATCC 1500 CTAAACAGAG TTCGGATTGA GATCCCATTA GAAAAGGAAA ACCAAGATAA GTTAGTGAAG 1560 TTCATGATCC GGATTGGTGT TTTCAGCATT CTCTACCTTG TGCCACTCTT GGTTGTAATT 1620 GGATGTTACT TTTATGAGCA AGCTTACCGC GGCATCTGGG AGACAACATG GATCCAGGAA 1680 CGCTGCAGAG AGTATCACAT TCCATGTCCG TACCAGGTTA CTCAGATGAG TCGTCCAGAC 1740 CTGATTCTCT TTCTGATGAA GTATCTCATG GCTCTCATAG TTGGGATTCC CTCTATATTT 1800 TGGGTTGGAA GCAAAAAGAC ATGCTTTGAA TGGGCCAGTT TTTTCCATGG GCGTAGGAAA 1860 AAAGAGATAG TGAATGAGAG CCGGCAGGTG CTCCAGGAAC CTGACTTTGC TCAGTCACTC 1920 CTGAGGGACC CAAATACTCC AATTATAAGA AAATCAAGAG GAACTTCCAC TCAAGGGACA 1980 TCCACACATG CTTCTTCAAC TCAGCTGGCC ATGGTGGATG ACCAAAGAAG CAAAGCAGGG 2040 AGTGTCCACA GCAAAGTGAG CAGCTACCAT GGCAGCCTCC ACAGGTCACG GGATGGCAGG 2100 TACACTCCCT GCAGTTACCG AGGAATGGAG GAGAGACTAC CTCACGGCAG CATGTCACGG 2160 CTGACGGATC ATTCCAGGCA CAGTAGTTCT CATCGGCTCA ACGAGCAGTC CCGACACAGC 2220 AGCATCCGAG ACCTCAGTAA CAACCCCATG ACTCACATTA CACATGGCAC CAGCATGAAC 2280 CGTGTTATTG AGGAGGATGG AACCAGTGCT TAGTCTTGTC TAAGGTGAAA TGTGTGCTGT 2340 TGAAAAGCAG GTTTTGCCTT CGCATGGCTG GCTGCTGTAA CTCACTGTCG CTCTGCTTTC 2400 TTGGGCAGAG TGTCAGCCTG GGAAAGTAGA TCTTTGCTCT TTGTATCACA TCAACCCTGG 2460 GGTGTGAACA CATCCAAACC CTAAGGATCA TGTCATCACA AAAGTAATTC TTTCTAGGCT 2520 GTGAAGAGAT GATTGTCTGG TGAGCATTTT TTATAAACAT GCTTATTTTA TATCTAGAAA 2580 AATCCTCTAT GTGTGGTGAC TGCTTTGTAG TGAATTTCAT ATAA 2624 667 amino acids amino acid single linear protein NO NO Mfz3 protein 4 Met Ala Val Ser Trp Ile Val Phe Asp Leu Trp Leu Leu Thr Val Phe 1 5 10 15 Leu Gly Gln Ile Gly Gly His Ser Leu Phe Ser Cys Glu Pro Ile Thr 20 25 30 Leu Arg Met Cys Gln Asp Leu Pro Tyr Asn Thr Thr Phe Met Pro Asn 35 40 45 Leu Leu Asn His Tyr Asp Gln Gln Thr Ala Ala Leu Ala Met Glu Pro 50 55 60 Phe His Pro Met Val Asn Leu Asp Cys Ser Arg Asp Phe Arg Pro Phe 65 70 75 80 Leu Cys Ala Leu Tyr Ala Pro Ile Cys Met Glu Tyr Gly Arg Val Thr 85 90 95 Leu Pro Cys Arg Arg Leu Cys Gln Arg Ala Tyr Ser Glu Cys Ser Lys 100 105 110 Leu Met Glu Met Phe Gly Val Pro Trp Pro Glu Asp Met Glu Cys Ser 115 120 125 Arg Phe Pro Asp Cys Asp Glu Pro Tyr Pro Arg Leu Val Asp Leu Asn 130 135 140 Leu Val Gly Asp Pro Thr Glu Gly Ala Pro Val Ala Val Gln Arg Asp 145 150 155 160 Tyr Gly Phe Trp Cys Pro Arg Glu Leu Lys Ile Asp Pro Asp Leu Gly 165 170 175 Tyr Ser Phe Leu His Val Arg Asp Cys Ser Pro Pro Cys Pro Asn Met 180 185 190 Tyr Phe Arg Arg Glu Glu Leu Ser Phe Ala Arg Tyr Phe Ile Gly Leu 195 200 205 Ile Ser Ile Ile Cys Leu Ser Ala Thr Leu Phe Thr Phe Leu Thr Phe 210 215 220 Leu Ile Asp Val Thr Arg Phe Arg Tyr Pro Glu Arg Pro Ile Ile Phe 225 230 235 240 Tyr Ala Val Cys Tyr Met Met Val Ser Leu Ile Phe Phe Ile Gly Phe 245 250 255 Leu Leu Glu Asp Arg Val Ala Cys Asn Ala Ser Ser Pro Ala Gln Tyr 260 265 270 Lys Ala Ser Thr Val Thr Gln Gly Ser His Asn Lys Ala Cys Thr Met 275 280 285 Leu Phe Met Val Leu Tyr Phe Phe Thr Met Ala Gly Ser Val Trp Trp 290 295 300 Val Ile Leu Thr Ile Thr Trp Phe Leu Ala Ala Val Pro Lys Trp Gly 305 310 315 320 Ser Glu Ala Ile Glu Lys Lys Ala Leu Leu Phe His Ala Ser Ala Trp 325 330 335 Gly Ile Pro Gly Thr Leu Thr Ile Ile Leu Leu Ala Met Asn Lys Ile 340 345 350 Glu Gly Asp Asn Ile Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Val 355 360 365 Asp Ala Leu Arg Tyr Phe Val Leu Ala Pro Leu Cys Leu Tyr Val Val 370 375 380 Val Gly Val Ser Leu Leu Leu Ala Gly Ile Ile Ser Leu Asn Arg Val 385 390 395 400 Arg Ile Glu Ile Pro Leu Glu Lys Glu Asn Gln Asp Lys Leu Val Lys 405 410 415 Phe Met Ile Arg Ile Gly Val Phe Ser Ile Leu Tyr Leu Val Pro Leu 420 425 430 Leu Val Val Ile Gly Cys Tyr Phe Tyr Glu Gln Ala Tyr Arg Gly Ile 435 440 445 Trp Glu Thr Thr Trp Ile Gln Glu Arg Cys Arg Glu Tyr His Ile Pro 450 455 460 Cys Pro Tyr Gln Val Thr Gln Met Ser Arg Pro Asp Leu Ile Leu Phe 465 470 475 480 Leu Met Lys Tyr Leu Met Ala Leu Ile Val Gly Ile Pro Ser Ile Phe 485 490 495 Trp Val Gly Ser Lys Lys Thr Cys Phe Glu Trp Ala Ser Phe Phe His 500 505 510 Gly Arg Arg Lys Lys Glu Ile Val Asn Glu Ser Arg Gln Val Leu Gln 515 520 525 Glu Pro Asp Phe Ala Gln Ser Leu Leu Arg Asp Pro Asn Thr Pro Ile 530 535 540 Ile Arg Lys Ser Arg Gly Thr Ser Thr Gln Gly Thr Ser Thr His Ala 545 550 555 560 Ser Ser Thr Gln Leu Ala Met Val Asp Asp Gln Arg Ser Lys Ala Gly 565 570 575 Ser Val His Ser Lys Val Ser Ser Tyr His Gly Ser Leu His Arg Ser 580 585 590 Arg Asp Gly Arg Tyr Thr Pro Cys Ser Tyr Arg Gly Met Glu Glu Arg 595 600 605 Leu Pro His Gly Ser Met Ser Arg Leu Thr Asp His Ser Arg His Ser 610 615 620 Ser Ser His Arg Leu Asn Glu Gln Ser Arg His Ser Ser Ile Arg Asp 625 630 635 640 Leu Ser Asn Asn Pro Met Thr His Ile Thr His Gly Thr Ser Met Asn 645 650 655 Arg Val Ile Glu Glu Asp Gly Thr Ser Ala Glx 660 665 1770 base pairs nucleic acid double linear DNA (genomic) NO NO Caenorhabditis elegans putative transmembrane receptor (frizzled 1) gene, Coding region 57..1634 5 GAATTCGGTT TAATTACCCA AGTTTGAGCT GTGAGCCCCC AATTCCATTA TCATTAATGG 60 GACCATTTCG TGGTTACCTC GGAGTAACCT GGCTCCTGTT GCTCTTTGTG ATTGGTGTGG 120 ACGGGCAGAG GTGTCAAAAG GTGGATCATG AGATGTGCAA CGATTTGCCG TATAACTTAA 180 CGAGCTTCCC AAATCTCGTC GACGAGGAAT CATGGAAAGA CGCCTCCGAA TCCATCCTCA 240 CCTACAAGCC CCTGCTCTCC GTTGTCTGCT CCGAGCAGCT CAAATTCTTC CTGTGCTCCG 300 TCTACTTCCC GATGTGCAAC GAGAAACTAG CCAACCCAAT TGGTCCATGC CGTCCATTGT 360 GTCTTTCCGT CCAGGAAAAG TGTCTTCCAG TGCTGGAAAG TTTCGGTTTC AAGTGGCCCG 420 ATGTGATTCG TTGTGATAAG TTCCCGTTGG AGAACAATCG AGAGAAAATG TGCATGAAAG 480 GGCCAAATGA GCAAGGAGCA ATTCAAGATG AGAGGGCAAA GTTTGCAGCG AAAGAAAGTG 540 AGGACGACGG TAATGATCGA GTAGAAGATA TTCAACGGGA GGTCGACCGC CTCAACGGAA 600 AATGCCCACA GGATGAGGTG TTCCTGAATC GATCCTCAAA GTGTGTGCCT TTGTGCTCGA 660 ACCCACAGAA GGTTGGGCAG ACTGACCGTG AATCCGCCAC CCGACTCTTG TTGTTTCTCT 720 CGCTGAGCTC TGTAATACTA ACAATTCTAT CAGTCTTCAT AGTCGGCTTA TCACGTCTCG 780 AGATGCTCCA CTCACTTACG GAAACTGCCA TGTTCTTCTC GTGCATCTCG TTTTGTGCGA 840 CATCGGTTAT TTATATTGTG AGCATTTCGT TTAAAGATCA GTTCCAAATC TCGTGCACCG 900 ACTACACCCA TCACCTGCTC TTCGTCGTCG GAGGGCTTTC CCATGTTCCA TGTTCTTCAG 960 TGGCCTCACT GATTTACTAC ACGGCAACTT GCTCACGTCT CTGGTGGCTC TTGATCTGTG 1020 TGTCGTGGAA TAAGGCGACA AGGACATCGC ATATATTGGA CGACTCCAGA ACCCGCGTGA 1080 TCATGCTCAT CCTGGGAATC CCGCTGGCTC CACTAATGCT CGCGCTACTC GCAAAAGCCG 1140 TCGCCGCCAA TCCCCTCACC GGACTCTGCT TCATCGGAGC AGCAAGCCCG GGCACCGACT 1200 GGATCTTCAA CTTCTGCCGG GAGCTCATTC TATTCCTCAT CAGCTCCATT GCTCTTTCGT 1260 CTGCTTGCTG CCGGCTTCTG GGCTCTGATG AGCAGGATGT CAATGGGTTT GCCGGAGTCA 1320 TTGCGGCAGT CTATCCGATT GCTGGACTAT TCTACATGCT TTCATTTGTG AACGATGCCA 1380 CCCAACCGTT TCTCTCACTT GACAGAAGTT TCAATGCGGT CTCGGCGACC AAGTTCTCGT 1440 TTGATCTACT TTTGAGCTTC ATCATGTGCG CGTTTTGTCT TATTTACTTG CTGTTCAAGC 1500 TGACTAGATC CTCATCAAAA GTTAGCAAAG AAGGATATCA ACCGGCGGTG CCGAAACTCC 1560 CGCAACCGGC AATTCCCGGC AGTGTACGTT CGAACACCTA CGCGTCGACG TTTCGAACTA 1620 ATAATATGAT TTGAAGGATT TTCAATAATT TTTTGTGAAA AACAACGGGT TTATATAGAT 1680 AGAAAACAAA AAGGTGGTCT CAATTTTTTT TCCGTGAAAA TAAATTTTTA TTGATTTTTA 1740 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1770 526 amino acids amino acid single linear protein NO NO Cfz1 protein 6 Met Gly Pro Phe Arg Gly Tyr Leu Gly Val Thr Trp Leu Leu Leu Leu 1 5 10 15 Phe Val Ile Gly Val Asp Gly Gln Arg Cys Gln Lys Val Asp His Glu 20 25 30 Met Cys Asn Asp Leu Pro Tyr Asn Leu Thr Ser Phe Pro Asn Leu Val 35 40 45 Asp Glu Glu Ser Trp Lys Asp Ala Ser Glu Ser Ile Leu Thr Tyr Lys 50 55 60 Pro Leu Leu Ser Val Val Cys Ser Glu Gln Leu Lys Phe Phe Leu Cys 65 70 75 80 Ser Val Tyr Phe Pro Met Cys Asn Glu Lys Leu Ala Asn Pro Ile Gly 85 90 95 Pro Cys Arg Pro Leu Cys Leu Ser Val Gln Glu Lys Cys Leu Pro Val 100 105 110 Leu Glu Ser Phe Gly Phe Lys Trp Pro Asp Val Ile Arg Cys Asp Lys 115 120 125 Phe Pro Leu Glu Asn Asn Arg Glu Lys Met Cys Met Lys Gly Pro Asn 130 135 140 Glu Gln Gly Ala Ile Gln Asp Glu Arg Ala Lys Phe Ala Ala Lys Glu 145 150 155 160 Ser Glu Asp Asp Gly Asn Asp Arg Val Glu Asp Ile Gln Arg Glu Val 165 170 175 Asp Arg Leu Asn Gly Lys Cys Pro Gln Asp Glu Val Phe Leu Asn Arg 180 185 190 Ser Ser Lys Cys Val Pro Leu Cys Ser Asn Pro Gln Lys Val Gly Gln 195 200 205 Thr Asp Arg Glu Ser Ala Thr Arg Leu Leu Leu Phe Leu Ser Leu Ser 210 215 220 Ser Val Ile Leu Thr Ile Leu Ser Val Phe Ile Val Gly Leu Ser Arg 225 230 235 240 Leu Glu Met Leu His Ser Leu Thr Glu Thr Ala Met Phe Phe Ser Cys 245 250 255 Ile Ser Phe Cys Ala Thr Ser Val Ile Tyr Ile Val Ser Ile Ser Phe 260 265 270 Lys Asp Gln Phe Gln Ile Ser Cys Thr Asp Tyr Thr His His Leu Leu 275 280 285 Phe Val Val Gly Gly Leu Ser His Val Pro Cys Ser Ser Val Ala Ser 290 295 300 Leu Ile Tyr Tyr Thr Ala Thr Cys Ser Arg Leu Trp Trp Leu Leu Ile 305 310 315 320 Cys Val Ser Trp Asn Lys Ala Thr Arg Thr Ser His Ile Leu Asp Asp 325 330 335 Ser Arg Thr Arg Val Ile Met Leu Ile Leu Gly Ile Pro Leu Ala Pro 340 345 350 Leu Met Leu Ala Leu Leu Ala Lys Ala Val Ala Ala Asn Pro Leu Thr 355 360 365 Gly Leu Cys Phe Ile Gly Ala Ala Ser Pro Gly Thr Asp Trp Ile Phe 370 375 380 Asn Phe Cys Arg Glu Leu Ile Leu Phe Leu Ile Ser Ser Ile Ala Leu 385 390 395 400 Ser Ser Ala Cys Cys Arg Leu Leu Gly Ser Asp Glu Gln Asp Val Asn 405 410 415 Gly Phe Ala Gly Val Ile Ala Ala Val Tyr Pro Ile Ala Gly Leu Phe 420 425 430 Tyr Met Leu Ser Phe Val Asn Asp Ala Thr Gln Pro Phe Leu Ser Leu 435 440 445 Asp Arg Ser Phe Asn Ala Val Ser Ala Thr Lys Phe Ser Phe Asp Leu 450 455 460 Leu Leu Ser Phe Ile Met Cys Ala Phe Cys Leu Ile Tyr Leu Leu Phe 465 470 475 480 Lys Leu Thr Arg Ser Ser Ser Lys Val Ser Lys Glu Gly Tyr Gln Pro 485 490 495 Ala Val Pro Lys Leu Pro Gln Pro Ala Ile Pro Gly Ser Val Arg Ser 500 505 510 Asn Thr Tyr Ala Ser Thr Phe Arg Thr Asn Asn Met Ile Glx 515 520 525 2828 base pairs nucleic acid double linear mRNA NO NO Mus musculus putative transmembrane receptor (frizzled 4) mRNA, Coding region 238..1941 7 TCGACCTCAA CACAAAGACC TGGGTCGTGA GACACACGCG TAGAGTCAGG CGGCTTCCCC 60 GAAAACCGGA CTCGGCCGGC GCCGAGTCTG GGTCCCCGCC TTCAACCATG ACCCTAGCAA 120 TCCATCCCTC GGCCCGGGCT CCGGACGTCT GATATTCCGC ACATTCTCGT ACAACTGCTG 180 GAGAGGCGAC TGCTGCCCCC TTGTCGCCCT TGGCGCCTTA CCGCATTCCC TATCCGGAGT 240 TGGGAGCAGC GCGGCCACCG GCGCCCCTGT GCAAACTGGG GGTGTCTGCT AGATCAGCCT 300 CTGCCGCTGC TGCCCGCAGC TCTGGCCATG GCCTGGCCGG GCACAGGGCC GAGCAGCCGG 360 GGGGCGCCTG GAGGCGTCGG GCTCAGGCTG GGGCTGCTGC TGCAGTTCCT CCTGCTCCTG 420 CGGCCGACAC TGGGGTTCGG GGACGAGGAG GAGCGGCGCT GCGACCCCAT CCGCATCGCC 480 ATGTGCCAGA ACCTCGGCTA CAACGTGACC AAGATGCCCA ACTTAGTGGG ACACGAGCTG 540 CAGACAGACG CCGAGCTGCA GCTGACAACT TTCACGCCGC TCATCCAGTA CGGCTGCTCC 600 AGCCAGCTGC AGTTCTTCCT TTGTTCGGTT TATGTGCCAA TGTGCACAGA GAAGATCAAC 660 ATCCCCATCG GCCCGTGCGG TGGCATGTGC CTTTCAGTCA AGAGACGCTG TGAACCAGTC 720 CTGAGAGAAT TTGGGTTTGC CTGGCCCGAC ACCCTGAACT GCAGCAAGTT CCCGCCCCAG 780 AACGACCACA ACCACATGTG CATGGAAGGA CCAGGTGATG AAGAGGTTCC CTTGCCCCAC 840 AAGACTCCCA TCCAGCCCGG GGAAGAGTGC CACTCCGTGG GAAGCAATTC TGATCAGTAC 900 ATCTGGGTGA AGAGGAGCCT GAACTGTGTT CTCAAGTGTG GCTACGATGC TGGCTTGTAC 960 AGCCGCTCAG CTAAGGAGTT CACGGATATT TGGATGGCTG TGTGGGCCAG CCTCTGCTTC 1020 ATCTCCACCA CCTTCACCGT GCTGACCTTC CTGATTGATT CATCCAGGTT TTCTTACCCT 1080 GAGCGCCCCA TCATATTTCT CAGTATGTGC TATAATATTT ATAGCATTGC TTATATTGTT 1140 CGGCTGACTG TAGGCCGGGA AAGGATATCC TGTGATTTTG AAGAGGCGGC AGAGCCCGTT 1200 CTCATCCAAG AAGGACTTAA GAACACAGGA TGTGCAATAA TTTTCTTGCT GATGTACTTT 1260 TTTGGAATGG CCAGCTCCAT TTGGTGGGTT ATTCTGACAC TCACTTGGTT TTTGGCAGCC 1320 GGACTCAAGT GGGGTCATGA AGCCATTGAA ATGCACAGTT CTTATTTCCA CATCGCAGCC 1380 TGGGCTATTC CCGCAGTGAA AACCATTGTC ATCTTGATTA TGAGACTAGT GGATGCCGAT 1440 GAACTGACTG GCTTGTGCTA TGTTGGGAAC CAAAACCTAG ATGCCCTCAC TGGCTTTGTG 1500 GTGGCTCCTC TCTTTACGTA TTTGGTGATT GGAACGCTGT TCATTGCGGC GGGTTTGGTG 1560 GCCTTATTCA AAATTCGGTC CAATCTTCAA AAAGACGGGA CAAAGACAGA CAAGTTGGAA 1620 AGGCTAATGG TCAAGATCGG GGTCTTCTCA GTACTGTACA CGGTTCCTGC AACCTGTGTG 1680 ATTGCCTGTT ATTTCTATGA AATCTCAAAC TGGGCACTCT TTCGATATTC TGCAGATGAC 1740 TCAAACATGG CAGTTGAAAT GTTGAAAATT TTTATGTCTT TGCTCGTGGG CATCACTTCA 1800 GGCATGTGGA TTTGGTCTGC CAAAACTCTT CACACGTGGC AAAAGTGTTC TAACCGATTG 1860 GTGAATTCTG GGAAGGTAAA GAGAGAGAAG AGGGGGAATG GTTGGGTGAA GCCAGGAAAA 1920 GGCAACGAGA CTGTGGTATA AGACTAGCCG GCTTCCTCGT TCCTCATTGT GAAGGAAGTG 1980 ATGCAGGGAA TCTCAGTTTG AACAAACTTA GAAACACTTC AGCCCACACA CACCCACGTC 2040 AGCCCACCAC CACTCACCCA ACTCAGCATC AGAAGACCAA TGGCTTCACT GCAGACTTTG 2100 GAATGGTCCA AAATGGAAAA GCCAGTTAAG AGGTTTTCAA AGCTGTGAAA AATCAAAATG 2160 TTGATCACTT TAGCAGGTCA CAGCTTGGAG TCCGTGGAGG TCCCGCCTAG ATTCCTGAAG 2220 CCCAGGGTGA TAGTGTTTGC TCCTACTGGG TGGGATTTCA ACTGTGAGTT GATAACATGC 2280 AAGGAGAAAG ATTAATTTTT AAAACCCTTT TAAATTTTAA ATAGTAACTA AGGTCTTGCA 2340 GATAGCAAAG TGATCTATAA ACACTGGAAA TGCTGGGTTG GGAGACGTGT TGCAGAGTTT 2400 TTATATGTTT CTGGTCTAAC ATAAACATCT TCTGGCCTAC ACTGTCTGCT GTTTAGAACT 2460 CTGTAGCGCA CTCCCAGAGG TGGTGTCAAA ATCCTTCAGT GCCTTGTCGT AAAACAGAAT 2520 TGTTTGAGCA AACAAAAGTA CTGTACTAAC ACACGTAAGG TATCCAGTGG ATTTCTCTCT 2580 CCTGAAATTT CAACATCCCT AATTCTAGGC AGCCCCTGTT TTCTTCACTT TAAACTAATG 2640 ACTCAAAAAA AAAAAGGTTA TTTTTATAGG ATTTTTTTTT GCACTGCAGC ATGCCTAATG 2700 AGAGGAAAAG GAGGTGATCA CTTCTGACAA TCACTTAATT CAGAGAAAAA TGAGATTTGC 2760 TAATTGACTT ACCTTCCGAC CCCTAGAGAC CCTATTGCAT TAAGCAATGT TTAAGCAATT 2820 GGGGACTT 2828 538 amino acids amino acid single linear protein NO NO Mfz4 protein 8 Met Ala Trp Pro Gly Thr Gly Pro Ser Ser Arg Gly Ala Pro Gly Gly 1 5 10 15 Val Gly Leu Arg Leu Gly Leu Leu Leu Gln Phe Leu Leu Leu Leu Arg 20 25 30 Pro Thr Leu Gly Phe Gly Asp Glu Glu Glu Arg Arg Cys Asp Pro Ile 35 40 45 Arg Ile Ala Met Cys Gln Asn Leu Gly Tyr Asn Val Thr Lys Met Pro 50 55 60 Asn Leu Val Gly His Glu Leu Gln Thr Asp Ala Glu Leu Gln Leu Thr 65 70 75 80 Thr Phe Thr Pro Leu Ile Gln Tyr Gly Cys Ser Ser Gln Leu Gln Phe 85 90 95 Phe Leu Cys Ser Val Tyr Val Pro Met Cys Thr Glu Lys Ile Asn Ile 100 105 110 Pro Ile Gly Pro Cys Gly Gly Met Cys Leu Ser Val Lys Arg Arg Cys 115 120 125 Glu Pro Val Leu Arg Glu Phe Gly Phe Ala Trp Pro Asp Thr Leu Asn 130 135 140 Cys Ser Lys Phe Pro Pro Gln Asn Asp His Asn His Met Cys Met Glu 145 150 155 160 Gly Pro Gly Asp Glu Glu Val Pro Leu Pro His Lys Thr Pro Ile Gln 165 170 175 Pro Gly Glu Glu Cys His Ser Val Gly Ser Asn Ser Asp Gln Tyr Ile 180 185 190 Trp Val Lys Arg Ser Leu Asn Cys Val Leu Lys Cys Gly Tyr Asp Ala 195 200 205 Gly Leu Tyr Ser Arg Ser Ala Lys Glu Phe Thr Asp Ile Trp Met Ala 210 215 220 Val Trp Ala Ser Leu Cys Phe Ile Ser Thr Thr Phe Thr Val Leu Thr 225 230 235 240 Phe Leu Ile Asp Ser Ser Arg Phe Ser Tyr Pro Glu Arg Pro Ile Ile 245 250 255 Phe Leu Ser Met Cys Tyr Asn Ile Tyr Ser Ile Ala Tyr Ile Val Arg 260 265 270 Leu Thr Val Gly Arg Glu Arg Ile Ser Cys Asp Phe Glu Glu Ala Ala 275 280 285 Glu Pro Val Leu Ile Gln Glu Gly Leu Lys Asn Thr Gly Cys Ala Ile 290 295 300 Ile Phe Leu Leu Met Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp 305 310 315 320 Val Ile Leu Thr Leu Thr Trp Phe Leu Ala Ala Gly Leu Lys Trp Gly 325 330 335 His Glu Ala Ile Glu Met His Ser Ser Tyr Phe His Ile Ala Ala Trp 340 345 350 Ala Ile Pro Ala Val Lys Thr Ile Val Ile Leu Ile Met Arg Leu Val 355 360 365 Asp Ala Asp Glu Leu Thr Gly Leu Cys Tyr Val Gly Asn Gln Asn Leu 370 375 380 Asp Ala Leu Thr Gly Phe Val Val Ala Pro Leu Phe Thr Tyr Leu Val 385 390 395 400 Ile Gly Thr Leu Phe Ile Ala Ala Gly Leu Val Ala Leu Phe Lys Ile 405 410 415 Arg Ser Asn Leu Gln Lys Asp Gly Thr Lys Thr Asp Lys Leu Glu Arg 420 425 430 Leu Met Val Lys Ile Gly Val Phe Ser Val Leu Tyr Thr Val Pro Ala 435 440 445 Thr Cys Val Ile Ala Cys Tyr Phe Tyr Glu Ile Ser Asn Trp Ala Leu 450 455 460 Phe Arg Tyr Ser Ala Asp Asp Ser Asn Met Ala Val Glu Met Leu Lys 465 470 475 480 Ile Phe Met Ser Leu Leu Val Gly Ile Thr Ser Gly Met Trp Ile Trp 485 490 495 Ser Ala Lys Thr Leu His Thr Trp Gln Lys Cys Ser Asn Arg Leu Val 500 505 510 Asn Ser Gly Lys Val Lys Arg Glu Lys Arg Gly Asn Gly Trp Val Lys 515 520 525 Pro Gly Lys Gly Asn Glu Thr Val Val Glx 530 535 2334 base pairs nucleic acid double linear mRNA NO NO Human transmembrane receptor (frizzled 5) mRNA, Coding region 321..2078 9 ACCCAGGGAC GGAGGACCCA GGCTGGCTTG GGGACTGTCT GCTCTTCTCG GCGGGAGCCG 60 TGGAGAGTCC TTTCCCTGGA ATCCGAGCCC TAACCGTCTC TCCCCAGCCC TATCCGGCGA 120 GGAGCGGAGC GCTGCCAGCG GAGGCAGCGC CTTCCCGAAG CAGTTTATCT TTGGACGGTT 180 TTCTTTAAAG GAAAAACGAA CCAACAGGTT GCCAGCCCCG GCGCCACACA CGAGACGCCG 240 GAGGGAGAAG CCCCGGCCCG GATTCCTCTG CCTGTGTGCG TCCCTCGCGG GCTGCTGGAG 300 GCGAGGGGAG GGAGGGGGCG ATGGCTCGGC CTGACCCATC CGCGCCGCCC TCGCTGTTGC 360 TGCTGCTCCT GGCGCAGCTG GTGGGCCGGG CGGCCGCCGC GTCCAAGGCC CCGGTGTGCC 420 AGGAAATCAC GGTGCCCATG TGCCGCGGCA TCGGCTACAA CCTGACGCAC ATGCCCAACC 480 AGTTCAACCA CGACACGCAG GACGAGGCGG GCCTGGAGGT GCACCAGTTC TGGCCGCTGG 540 TGGAGATCCA ATGCTCGCCG GACCTGCGCT TCTTCCTATG CACTATGTAC ACGCCCATCT 600 GTCTGCCCGA CTACCACAAG CCGCTGCCGC CCTGCCGCTC GGTGTGCGAG CGCGCCAAGG 660 CCGGCTGCTC GCCGCTGATG CGCCAGTACG GCTTCGCCTG GCCCGAGCGC ATGAGCTGCG 720 ACCGCCTCCC GGTGCTGGGC CGCGACGCCG AGGTCCTCTG CATGGATTAC AACCGCAGCG 780 AGGCCACCAC GGCGCCCCCC AGGCCTTTCC CAGCCAAGCC CACCCTTCCA GGCCCGCCAG 840 GGGCGCCGGC CTCGGGGGGC GAATGCCCCG CTGGGGGCCC GTTCGTGTGC AAGTGTCGCG 900 AGCCCTTCGT GCCCATTCTG AAGGAGTCAC ACCCGCTCTA CAACAAGGTG CGGACGGGCC 960 AGGTGCCCAA CTGCGCGGTA CCCTGCTACC AGCCGTCCTT CAGTGCCGAC GAGCGCACGT 1020 TCGCCACCTT CTGGATAGGC CTGTGGTCGG TGCTGTGCTT CATCTCCACG TCCACCACAG 1080 TGGCCACCTT CCTCATCGAC ATGGACACGT TCCGCTATCC TGAGCGCCCC ATCATCTTCC 1140 TGTCAGCCTG CTACCTGTGC GTGTCGCTGG GCTTCCTGGT GCGTCTGGTC GTGGGCCATG 1200 CCAGCGTGGC CTGCAGCCGC GAGCACAACC ACATCCACTA CGAGACCACG GGCCCTGCAC 1260 TGTGCACCAT CGTCTTCCTC CTGGTCTACT TCTTCGGCAT GGCCAGCTCC ATCTGGTGGG 1320 TCATCCTGTC GCTCACCTGG TTCCTGGCCG CCGCGATGAA GTGGGGCAAC GAGGCCATCG 1380 CGGGCTACGG CCAGTACTTC CACCTGGCTG CGTGGCTCAT CCCCAGCGTC AAGTCCATCA 1440 CGGCACTGGC GCTGAGCTCC GTGGACGGGG ACCCAGTGGC CGGCATCTGC TACGTGGGCA 1500 ACCAGAACCT GAACTCGCTG CGGCGCTTCG TGCTGGGCCC GCTGGTGCTC TACCTGCTGG 1560 TGGGCACGCT CTTCCTGCTG GCGGGCTTCG TGTCGCTCTT CCGCATCCGC AGCGTCATCA 1620 AGCAGGGCGG CACCAAGACG GACAAGCTGG AGAAGCTCAT GATCCGCATC GGCATCTTCA 1680 CGCTGCTCTA CACGGTCCCC GCCAGCATTG TGGTGGCCTG CTACCTGTAC GAGCAGCACT 1740 ACCGCGAGAG CTGGGAGGCG GCGCTCACCT GCGCCTGCCC GGGCCACGAC ACCGGCCAGC 1800 CGCGCGCCAA GCCCGAGTAC TGGGTGCTCA TGCTCAAGTA CTTCATGTGC CTGGTGGTGG 1860 GCATCACGTC GGGCGTCTGG ATCTGGTCGG GCAAGACGGT GGAGTCGTGG CGGCGTTTCA 1920 CCAGCCGCTG CTGCTGCCGC CCGCGGCGCG GCCACAAGAG CGGGGGCGCC ATGGCCGCAG 1980 GGGACTACCC CGAGGCGAGC GCCGCGCTCA CAGGCAGGAC CGGGCCGCCG GGCCCCGCCG 2040 CCACCTACCA CAAGCAGGTG TCCCTGTCGC ACGTGTAGGA GGCTGCCGCC GAGGGACTCG 2100 GCCGGAGAGC TGAGGGGAGG GGGGCGTTTT GTTTGGTAGT TTTGCCAAGG TCACTTCCGT 2160 TTACCTTCAT GGTGCTGTTG CCCCCTCCCG CGGCGACTTG GAGAGAGGGA AGAGGGGCGT 2220 TTTCGAGGAA GAACCTGTCC CAGGTCTTCT CCAAGGGGCC CAGCTCACGT GTATTCTATT 2280 TTGCGTTTCT TACCTGCCTT CTTTATGGGA ACCCTCTTTT TAATTTATAT GTAT 2334 586 amino acids amino acid single linear protein NO NO Hfz5 protein 10 Met Ala Arg Pro Asp Pro Ser Ala Pro Pro Ser Leu Leu Leu Leu Leu 1 5 10 15 Leu Ala Gln Leu Val Gly Arg Ala Ala Ala Ala Ser Lys Ala Pro Val 20 25 30 Cys Gln Glu Ile Thr Val Pro Met Cys Arg Gly Ile Gly Tyr Asn Leu 35 40 45 Thr His Met Pro Asn Gln Phe Asn His Asp Thr Gln Asp Glu Ala Gly 50 55 60 Leu Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys Ser Pro 65 70 75 80 Asp Leu Arg Phe Phe Leu Cys Thr Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95 Asp Tyr His Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110 Lys Ala Gly Cys Ser Pro Leu Met Arg Gln Tyr Gly Phe Ala Trp Pro 115 120 125 Glu Arg Met Ser Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135 140 Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro 145 150 155 160 Arg Pro Phe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly Ala Pro 165 170 175 Ala Ser Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe Val Cys Lys Cys 180 185 190 Arg Glu Pro Phe Val Pro Ile Leu Lys Glu Ser His Pro Leu Tyr Asn 195 200 205 Lys Val Arg Thr Gly Gln Val Pro Asn Cys Ala Val Pro Cys Tyr Gln 210 215 220 Pro Ser Phe Ser Ala Asp Glu Arg Thr Phe Ala Thr Phe Trp Ile Gly 225 230 235 240 Leu Trp Ser Val Leu Cys Phe Ile Ser Thr Ser Thr Thr Val Ala Thr 245 250 255 Phe Leu Ile Asp Met Asp Thr Phe Arg Tyr Pro Glu Arg Pro Ile Ile 260 265 270 Phe Leu Ser Ala Cys Tyr Leu Cys Val Ser Leu Gly Phe Leu Val Arg 275 280 285 Leu Val Val Gly His Ala Ser Val Ala Cys Ser Arg Glu His Asn His 290 295 300 Ile His Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr Ile Val Phe Leu 305 310 315 320 Leu Val Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp Val Ile Leu 325 330 335 Ser Leu Thr Trp Phe Leu Ala Ala Ala Met Lys Trp Gly Asn Glu Ala 340 345 350 Ile Ala Gly Tyr Gly Gln Tyr Phe His Leu Ala Ala Trp Leu Ile Pro 355 360 365 Ser Val Lys Ser Ile Thr Ala Leu Ala Leu Ser Ser Val Asp Gly Asp 370 375 380 Pro Val Ala Gly Ile Cys Tyr Val Gly Asn Gln Asn Leu Asn Ser Leu 385 390 395 400 Arg Arg Phe Val Leu Gly Pro Leu Val Leu Tyr Leu Leu Val Gly Thr 405 410 415 Leu Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser Val 420 425 430 Ile Lys Gln Gly Gly Thr Lys Thr Asp Lys Leu Glu Lys Leu Met Ile 435 440 445 Arg Ile Gly Ile Phe Thr Leu Leu Tyr Thr Val Pro Ala Ser Ile Val 450 455 460 Val Ala Cys Tyr Leu Tyr Glu Gln His Tyr Arg Glu Ser Trp Glu Ala 465 470 475 480 Ala Leu Thr Cys Ala Cys Pro Gly His Asp Thr Gly Gln Pro Arg Ala 485 490 495 Lys Pro Glu Tyr Trp Val Leu Met Leu Lys Tyr Phe Met Cys Leu Val 500 505 510 Val Gly Ile Thr Ser Gly Val Trp Ile Trp Ser Gly Lys Thr Val Glu 515 520 525 Ser Trp Arg Arg Phe Thr Ser Arg Cys Cys Cys Arg Pro Arg Arg Gly 530 535 540 His Lys Ser Gly Gly Ala Met Ala Ala Gly Asp Tyr Pro Glu Ala Ser 545 550 555 560 Ala Ala Leu Thr Gly Arg Thr Gly Pro Pro Gly Pro Ala Ala Thr Tyr 565 570 575 His Lys Gln Val Ser Leu Ser His Val Glx 580 585 2492 base pairs nucleic acid double linear mRNA NO NO Mus musculus putative transmembrane receptor (frizzled 6) mRNA, Coding region 146..2275 11 TCATTTCAGG CCCAGCTACT ATCAAAATGG TACAAAGAAT GCAATGAGGA ATTTGTACAT 60 TTTATCTCTG ATTTGAGAAT CTTTTTGATG CGGAAAGGAG CATAAGAATA ATCCAAGCCA 120 TGTGGTAAAA TCGGAGTCTG GCAAGATGGA AAGGTCCCCG TTTCTGTTGG CGTGCATTCT 180 TCTGCCCCTC GTAAGAGGAC ACAGCCTTTT CACCTGTGAG CCAATCACCG TTCCCAGATG 240 TATGAAAATG ACTTACAACA TGACGTTCTT CCCTAACCTG ATGGGTCATT ATGACCAGGG 300 GATCGCTGCT GTGGAAATGG GGCACTTTCT GCATCTTGCA AATCTAGAAT GTTCACCAAA 360 CATTGAAATG TTCCTTTGCC AAGCTTTTAT ACCAACCTGC ACAGAGCAAA TTCATGTAGT 420 TCTACCCTGT CGGAAATTGT GTGAGAAAAT AGTTTCTGAT TGCAAAAAAC TAATGGACAC 480 TTTTGGCATC CGATGGCCTG AAGAACTTGA ATGTAACAGA TTGCCACACT GTGATGACAC 540 TGTTCCTGTA ACTTCTCATC CACACACAGA GCTTTCTGGG CCACAGAAGA AATCAGATCA 600 AGTCCCAAGA GACATTGGAT TTTGGTGTCC AAAGCACCTT AGGACTTCCG GGGACCAAGG 660 CTATAGGTTT CTGGGAATTG AACAGTGTGC CCCTCCGTGC CCCAATATGT ATTTTAAAAG 720 TGATGAACTA GACTTTGCCA AAAGTTTCAT AGGAATAGTT TCAATATTTT GTCTTTGTGC 780 AACTCTGTTC ACGTTCCTTA CATTTTTAAT TGACGTTAGA CGATTCAGAT ACCCAGAGAG 840 ACCAATTATC TATTACTCTG TCTGCTACAG CATTGTCTCT CTCATGTACT TCGTGGGGTT 900 TTTGCTGGGC AATAGCACAG CTTGTAATAA GGCAGACGAG AAGCTGGAGC TCGGGGACAC 960 CGTTGTCCTA GGGTCAAAGA ATAAGGCTTG CAGTGTGGTA TTTATGTTTC TGTATTTTTT 1020 TACAATGGCT GGCACCGTGT GGTGGGTGAT TCTCACCATT ACGTGGTTCT TAGCTGCCGG 1080 GAGAAAATGG AGTTGCGAAG CTATTGAACA AAAAGCAGTG TGGTTCCATG CCGTTGCCTG 1140 GGGGGCGCCC GGGTTCCTGA CCGTCATGCT GCTCGCTATG AATAAGGTTG AAGGAGACAA 1200 CATTAGCGGC GTTTGCTTCG TTGGCCTGTA TGACCTGGAC GCCTCTCGCT ACTTCGTCCT 1260 TCTGCCTCTG TGCCTCTGCG TATTTGTTGG GCTGTCTCTC CTCTTAGCCG GCATCATCTC 1320 CTTGAATCAT GTCCGACAAG TCATACAGCA TGATGGTCGG AACCAAGAGA AGCTAAAGAA 1380 ATTCATGATT CGCATCGGAG TCTTCAGTGG CCTGTATCTT GTGCCCTTAG TGACACTTCT 1440 CGGTTGCTAT GTCTATGAGC TAGTGAACAG GATCACCTGG GAGATGACAT GGTTCTCTGA 1500 TCATTGTCAC CAGTACCGCA TCCCGTGCCC TTACCAGGCA AATCCAAAAG CTCGACCAGA 1560 ATTGGCTTTA TTTATGATAA AATATCTGAT GACATTAATT GTTGGTATCT CTGCGGTCTT 1620 CTGGGTTGGA AGCAAAAAGA CGTGCACAGA ATGGGCCGGG TTCTTTAAGC GAAACCGCAA 1680 GCGAGACCCC ATCAGTGAGA GCCGCCGAGT GCTGCAAGAG TCCTGTGAGT TCTTCCTGAA 1740 GCACAACTCT AAAGTGAAGC ACAAGAAGAA GCATGGCGCA CCAGGGCCTC ATAGGCTGAA 1800 GGTCATTTCC AAGTCCATGG GAACTAGCAC AGGAGCGACC ACAAATCATG GCACCTCTGC 1860 CATGGCAATC GCTGACCATG ATTACTTAGG GCAAGAAACT TCAACAGAAG TCCACACCTC 1920 CCCAGAAGCA TCCGTCAAAG AGGGACGAGC AGACCGAGCA AACACTCCCA GCGCCAAAGA 1980 TCGGGACTGT GGGGAATCTG CAGGGCCCAG TTCCAAGCTC TCTGGGAACC GGAACGGCAG 2040 GGAAAGCCGA GCGGGCGGCC TGAAGGAGAG AAGCAATGGA TCAGAGGGGG CTCCAAGTGA 2100 AGGAAGGGTA AGTCCAAAGA GCAGCGTTCC TGAGACTGGC CTGATAGACT GCAGCACTTC 2160 ACAGGCCGCC AGTTCTCCAG AACCAACCAG CCTCAAGGGC TCCACATCTC TGCCTGTTCA 2220 CTCAGCTTCC AGAGCTAGGA AAGAGCAGGG TGCTGGCAGC CATTCCGACG CTTGAAGAAA 2280 ACTGTCTCGT TCCCCCAGAA GCACATGTAT GTTACACTGG AGATGACCAA CTGATTTGTC 2340 TTATAAAGGC CACTGTTGAG CTGGGAGAGT AGCCCAGTGG TACAGCGCCC ACCTGGAATA 2400 CTGAGGACCT GGGGTTGTCT CCCAGCACTG CAAAAGGAAA ATTCACTGTT ACAGTCTTCC 2460 TTGCACTTAA CCAGCTTTGT CTATGTTTTT TT 2492 710 amino acids amino acid single linear protein NO NO Mfz6 protein 12 Met Glu Arg Ser Pro Phe Leu Leu Ala Cys Ile Leu Leu Pro Leu Val 1 5 10 15 Arg Gly His Ser Leu Phe Thr Cys Glu Pro Ile Thr Val Pro Arg Cys 20 25 30 Met Lys Met Thr Tyr Asn Met Thr Phe Phe Pro Asn Leu Met Gly His 35 40 45 Tyr Asp Gln Gly Ile Ala Ala Val Glu Met Gly His Phe Leu His Leu 50 55 60 Ala Asn Leu Glu Cys Ser Pro Asn Ile Glu Met Phe Leu Cys Gln Ala 65 70 75 80 Phe Ile Pro Thr Cys Thr Glu Gln Ile His Val Val Leu Pro Cys Arg 85 90 95 Lys Leu Cys Glu Lys Ile Val Ser Asp Cys Lys Lys Leu Met Asp Thr 100 105 110 Phe Gly Ile Arg Trp Pro Glu Glu Leu Glu Cys Asn Arg Leu Pro His 115 120 125 Cys Asp Asp Thr Val Pro Val Thr Ser His Pro His Thr Glu Leu Ser 130 135 140 Gly Pro Gln Lys Lys Ser Asp Gln Val Pro Arg Asp Ile Gly Phe Trp 145 150 155 160 Cys Pro Lys His Leu Arg Thr Ser Gly Asp Gln Gly Tyr Arg Phe Leu 165 170 175 Gly Ile Glu Gln Cys Ala Pro Pro Cys Pro Asn Met Tyr Phe Lys Ser 180 185 190 Asp Glu Leu Asp Phe Ala Lys Ser Phe Ile Gly Ile Val Ser Ile Phe 195 200 205 Cys Leu Cys Ala Thr Leu Phe Thr Phe Leu Thr Phe Leu Ile Asp Val 210 215 220 Arg Arg Phe Arg Tyr Pro Glu Arg Pro Ile Ile Tyr Tyr Ser Val Cys 225 230 235 240 Tyr Ser Ile Val Ser Leu Met Tyr Phe Val Gly Phe Leu Leu Gly Asn 245 250 255 Ser Thr Ala Cys Asn Lys Ala Asp Glu Lys Leu Glu Leu Gly Asp Thr 260 265 270 Val Val Leu Gly Ser Lys Asn Lys Ala Cys Ser Val Val Phe Met Phe 275 280 285 Leu Tyr Phe Phe Thr Met Ala Gly Thr Val Trp Trp Val Ile Leu Thr 290 295 300 Ile Thr Trp Phe Leu Ala Ala Gly Arg Lys Trp Ser Cys Glu Ala Ile 305 310 315 320 Glu Gln Lys Ala Val Trp Phe His Ala Val Ala Trp Gly Ala Pro Gly 325 330 335 Phe Leu Thr Val Met Leu Leu Ala Met Asn Lys Val Glu Gly Asp Asn 340 345 350 Ile Ser Gly Val Cys Phe Val Gly Leu Tyr Asp Leu Asp Ala Ser Arg 355 360 365 Tyr Phe Val Leu Leu Pro Leu Cys Leu Cys Val Phe Val Gly Leu Ser 370 375 380 Leu Leu Leu Ala Gly Ile Ile Ser Leu Asn His Val Arg Gln Val Ile 385 390 395 400 Gln His Asp Gly Arg Asn Gln Glu Lys Leu Lys Lys Phe Met Ile Arg 405 410 415 Ile Gly Val Phe Ser Gly Leu Tyr Leu Val Pro Leu Val Thr Leu Leu 420 425 430 Gly Cys Tyr Val Tyr Glu Leu Val Asn Arg Ile Thr Trp Glu Met Thr 435 440 445 Trp Phe Ser Asp His Cys His Gln Tyr Arg Ile Pro Cys Pro Tyr Gln 450 455 460 Ala Asn Pro Lys Ala Arg Pro Glu Leu Ala Leu Phe Met Ile Lys Tyr 465 470 475 480 Leu Met Thr Leu Ile Val Gly Ile Ser Ala Val Phe Trp Val Gly Ser 485 490 495 Lys Lys Thr Cys Thr Glu Trp Ala Gly Phe Phe Lys Arg Asn Arg Lys 500 505 510 Arg Asp Pro Ile Ser Glu Ser Arg Arg Val Leu Gln Glu Ser Cys Glu 515 520 525 Phe Phe Leu Lys His Asn Ser Lys Val Lys His Lys Lys Lys His Gly 530 535 540 Ala Pro Gly Pro His Arg Leu Lys Val Ile Ser Lys Ser Met Gly Thr 545 550 555 560 Ser Thr Gly Ala Thr Thr Asn His Gly Thr Ser Ala Met Ala Ile Ala 565 570 575 Asp His Asp Tyr Leu Gly Gln Glu Thr Ser Thr Glu Val His Thr Ser 580 585 590 Pro Glu Ala Ser Val Lys Glu Gly Arg Ala Asp Arg Ala Asn Thr Pro 595 600 605 Ser Ala Lys Asp Arg Asp Cys Gly Glu Ser Ala Gly Pro Ser Ser Lys 610 615 620 Leu Ser Gly Asn Arg Asn Gly Arg Glu Ser Arg Ala Gly Gly Leu Lys 625 630 635 640 Glu Arg Ser Asn Gly Ser Glu Gly Ala Pro Ser Glu Gly Arg Val Ser 645 650 655 Pro Lys Ser Ser Val Pro Glu Thr Gly Leu Ile Asp Cys Ser Thr Ser 660 665 670 Gln Ala Ala Ser Ser Pro Glu Pro Thr Ser Leu Lys Gly Ser Thr Ser 675 680 685 Leu Pro Val His Ser Ala Ser Arg Ala Arg Lys Glu Gln Gly Ala Gly 690 695 700 Ser His Ser Asp Ala Glx 705 710 2259 base pairs nucleic acid double linear mRNA NO NO Mus musculus transmembrane receptor (frizzled 7) mRNA, Coding region 362..2080 13 TTTGAAGGTA ACCGGAGAAG CTTGTTGCTC GTCGCCGCAG AGAAAGCCGC ACCGTTACGT 60 CTCGGGGGGA GGGTAAGGCG ACACCCCTTC CCTCGTACCC CCACTCCAGG CCCAGGAGTT 120 TGAACTCCGG CGGCTGCGTG AGTGCCACGT GGAGGCGGCT GCGGCGCCCC TCGGCTGGCG 180 GCCTCGCCCC CGCTGTGCAG GCACCCTAGC ACCCTCGGCT CCGCGCCGCC CACGGCGGCC 240 CCGGCGCCGG GAGGACTCTC ATGCGCCGGC CGGGCGGCGG CGCCTCCCTG TATCCAAGCC 300 TCTCCCCAGC GCCTCGTCTT TTTCCTCCAG CTGAGAACGC CGCTGCACTC GCGACCGGCG 360 ATGCGGGGCC CCGGCACGGC GGCGTCGCAC TCGCCCCTGG GCCTCTGCGC CCTGGTGCTT 420 GCTCTTCTGG GCGCGCTGCC CACGGACACC CGGGCTCAGC CATATCACGG CGAGAAAGGC 480 ATCTCGGTAC CGGACCACGG CTTCTGCCAG CCCATCTCCA TCCCGTTGTG CACGGATATC 540 GCCTACAACC AGACCATCCT GCCCAACCTG CTGGGCCACA CGAACCAAGA GGACGCGGGC 600 CTCGAGGTGC ACCAGTTCTA CCCTCTGGTA AAGGTGCAGT GTTCTCCTGA GCTACGCTTC 660 TTCTTATGCT CTATGTACGC ACCCGTGTGC ACCGTGCTCG ACCAAGCCAT TCCTCCGTGC 720 CGTTCCTTGT GCGAGCGCGC CCGACAGGGC TGCGAGGCGC TCATGAACAA GTTCGGCTTC 780 CAGTGGCCAG AGCGGTTGCG CTGCGAGAAC TTCCCAGTGC ACGGTGCCGG CGAGATCTGC 840 GTGGGGCAGA ACACGTCCGA CGGCTCCGGG GGCGCGGGCG GCAGTCCCAC CGCCTACCCT 900 ACTGCTCCCT ACCTGCCAGA CCCACCTTTC ACTGCGATGT CCCCCTCAGA TGGCAGAGGC 960 CGCTTGTCTT TCCCCTTCTC GTGTCCGCGC CAGCTCAAGG TGCCCCCCTA CCTGGGCTAC 1020 CGCTTCCTAG GTGAGCGTGA CTGCGGTGCC CCGTGTGAGC CGGGCCGTGC TAACGGCCTC 1080 ATGTACTTTA AAGAAGAGGA GAGACGGTTC GCCCGCCTCT GGGTGGGTGT GTGGTCAGTG 1140 CTGTCGTGCG CCTCGACGCT CTTCACGGTG CTCACCTACC TAGTGGACAT GCGTCGCTTC 1200 AGCTATCCAG AGCGACCCAT CATCTTCCTG TCGGGTTGCT ACTTCATGGT GGCAGTGGCG 1260 CACGTGGCAG GCTTCCTGCT AGAGGACCGT GCCGTGTGCG TGGAGCGCTT CTCGGACGAT 1320 GGCTACCGCA CGGTGGCGCA GGGCACCAAG AAGGAGGGCT GCACCATCCT CTTCATGGTG 1380 CTTTACTTCT TCGGTATGGC CAGCTCCATC TGGTGGGTCA TTCTGTCCCT CACTTGGTTC 1440 CTGGCAGCTG GCATGAAGTG GGGCCACGAG GCCATCGAGG CCAACTCGCA GTACTTTCAT 1500 CTGGCCGCGT GGGCTGTGCC AGCGGTCAAG ACAATCACCA TTTTGGCCAT GGGCCAGGTG 1560 GATGGTGACC TACTCAGTGG AGTGTGCTAC GTGGGCCTGT CTAGTGTGGA TGCATTGCGG 1620 GGCTTCGTGC TGGCGCCCTT GTTCGTCTAC CTCTTCATCG GGACGTCCTT CCTGTTGGCC 1680 GGCTTTGTGT CTCTCTTTCG CATCCGCACC ATCATGAAGC ACGACGGCAC CAAGACAGAG 1740 AAGCTGGAGA AGCTGATGGT GCGCATCGGC GTCTTCAGCG TGCTCTACAC GGTGCCGGCC 1800 ACCATCGTGT TGGCCTGCTA CTTTTATGAG CAGGCCTTCC GAGAGCACTG GGAACGCACC 1860 TGGCTCCTGC AGACTTGCAA GAGCTACGCT GTGCCCTGCC CTCCGCGCCA CTTCTCTCCC 1920 ATGAGCCCCG ACTTTACAGT CTTCATGATC AAGTACCTGA TGACCATGAT CGTGGGCATC 1980 ACTACGGGCT TCTGGATCTG GTCGGGCAAG ACCCTGCAGT CATGGCGTCG CTTCTACCAC 2040 AGACTCAGCC ACAGCAGCAA GGGGGAAACT GCGGTATGAG CCCCGGTCCT TACCCACCCT 2100 TGCCTCTTCT ACCCTTTTAC AGGAGGAGAG GCATGGTAGG GAGAGAACTG CTGGGTGGGG 2160 GCTTGTTTCC GTAAGCTACC TGCCCCCTCC ACTGAGCTTT AACCTGGAAG TGAGAAGTTA 2220 TTTGGAGGTG AGAAGAGATT TGGGGGCGAG AGATGGTTT 2259 573 amino acids amino acid single linear protein NO NO Mfz7 protein 14 Met Arg Gly Pro Gly Thr Ala Ala Ser His Ser Pro Leu Gly Leu Cys 1 5 10 15 Ala Leu Val Leu Ala Leu Leu Gly Ala Leu Pro Thr Asp Thr Arg Ala 20 25 30 Gln Pro Tyr His Gly Glu Lys Gly Ile Ser Val Pro Asp His Gly Phe 35 40 45 Cys Gln Pro Ile Ser Ile Pro Leu Cys Thr Asp Ile Ala Tyr Asn Gln 50 55 60 Thr Ile Leu Pro Asn Leu Leu Gly His Thr Asn Gln Glu Asp Ala Gly 65 70 75 80 Leu Glu Val His Gln Phe Tyr Pro Leu Val Lys Val Gln Cys Ser Pro 85 90 95 Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110 Leu Asp Gln Ala Ile Pro Pro Cys Arg Ser Leu Cys Glu Arg Ala Arg 115 120 125 Gln Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gln Trp Pro Glu 130 135 140 Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu Ile Cys 145 150 155 160 Val Gly Gln Asn Thr Ser Asp Gly Ser Gly Gly Ala Gly Gly Ser Pro 165 170 175 Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Pro Pro Phe Thr Ala 180 185 190 Met Ser Pro Ser Asp Gly Arg Gly Arg Leu Ser Phe Pro Phe Ser Cys 195 200 205 Pro Arg Gln Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe Leu Gly 210 215 220 Glu Arg Asp Cys Gly Ala Pro Cys Glu Pro Gly Arg Ala Asn Gly Leu 225 230 235 240 Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu Trp Val Gly 245 250 255 Val Trp Ser Val Leu Ser Cys Ala Ser Thr Leu Phe Thr Val Leu Thr 260 265 270 Tyr Leu Val Asp Met Arg Arg Phe Ser Tyr Pro Glu Arg Pro Ile Ile 275 280 285 Phe Leu Ser Gly Cys Tyr Phe Met Val Ala Val Ala His Val Ala Gly 290 295 300 Phe Leu Leu Glu Asp Arg Ala Val Cys Val Glu Arg Phe Ser Asp Asp 305 310 315 320 Gly Tyr Arg Thr Val Ala Gln Gly Thr Lys Lys Glu Gly Cys Thr Ile 325 330 335 Leu Phe Met Val Leu Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp 340 345 350 Val Ile Leu Ser Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly 355 360 365 His Glu Ala Ile Glu Ala Asn Ser Gln Tyr Phe His Leu Ala Ala Trp 370 375 380 Ala Val Pro Ala Val Lys Thr Ile Thr Ile Leu Ala Met Gly Gln Val 385 390 395 400 Asp Gly Asp Leu Leu Ser Gly Val Cys Tyr Val Gly Leu Ser Ser Val 405 410 415 Asp Ala Leu Arg Gly Phe Val Leu Ala Pro Leu Phe Val Tyr Leu Phe 420 425 430 Ile Gly Thr Ser Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile 435 440 445 Arg Thr Ile Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu Glu Lys 450 455 460 Leu Met Val Arg Ile Gly Val Phe Ser Val Leu Tyr Thr Val Pro Ala 465 470 475 480 Thr Ile Val Leu Ala Cys Tyr Phe Tyr Glu Gln Ala Phe Arg Glu His 485 490 495 Trp Glu Arg Thr Trp Leu Leu Gln Thr Cys Lys Ser Tyr Ala Val Pro 500 505 510 Cys Pro Pro Arg His Phe Ser Pro Met Ser Pro Asp Phe Thr Val Phe 515 520 525 Met Ile Lys Tyr Leu Met Thr Met Ile Val Gly Ile Thr Thr Gly Phe 530 535 540 Trp Ile Trp Ser Gly Lys Thr Leu Gln Ser Trp Arg Arg Phe Tyr His 545 550 555 560 Arg Leu Ser His Ser Ser Lys Gly Glu Thr Ala Val Glx 565 570 2421 base pairs nucleic acid double linear DNA (genomic) NO NO Mus musculus transmembrane receptor (frizzled 8) gene, Coding region 188..2245 15 GGGGGAGGGC CGGACGACTC CAGCCTAGGT TTCCAACCCT GCTGCCTGAA AAGGAGATAG 60 ACTGTTGCTA TTCTCCTCTG CAGAGAAAAG TGGGACACGA CCCGCTCTCC CTTTTCTCAG 120 ATTCCTCACT GCAGAGCCCT CCTGCGCGCC GCCTAGAGAA GGAGGACTTG GGGTCCCAGC 180 GCGCAGCATG GAGTGGGGTT ACCTGTTGGA AGTGACCTCG CTCCTAGCCG CCTTGGCGGT 240 GCTACAGCGC TCTAGCGGCG CTGCCGCGGC TTCGGCCAAG GAGCTGGCGT GCCAAGAGAT 300 CACGGTGCCG TTGTGCAAAG GCATCGGTTA CAACTACACT TACATGCCCA ACCAGTTCAA 360 CCACGACACG CAAGATGAGG CGGGCCTAGA GGTGCACCAG TTTTGGCCGC TGGTGGAGAT 420 ACAGTGCTCC CCGGACCTCA AGTTCTTTCT GTGTAGCATG TACACGCCCA TCTGCCTGGA 480 GGACTACAAG AAGCCTCTGC CGCCTTGTCG CTCTGTGTGT GAACGCGCCA AGGCCGGCTG 540 CGCGCCGCTC ATGCGCCAGT ACGGCTTTGC TTGGCCTGAC CGCATGCGCT GCGATCGGTT 600 GCCGGAGCAG GGCAACCCGG ACACTCTGTG CATGGACTAC AACCGCACCG ACCTCACCAC 660 GGCCGCGCCC AGCCCACCGC GCCGCCTGCC TCCGCCGCCT CCTCCCGGCG AGCAGCCGCC 720 CTCTGGCAGC GGCCACAGCC GCCCGCCAGG GGCCAGGCCC CCACATCGTG GCGGCAGCAG 780 TAGGGGCAGC GGGGACGCGG CGGCTGCGCC CCCTTCGCGC GGCGGGAAGG CGAGGCCCCC 840 TGGTGGCGGC GCTGCTCCCT GCGAGCCGGG GTGCCAGTGC CGCGCGCCCA TGGTGAGCGT 900 GTCCAGCGAA CGCCACCCGC TCTACAACCG CGTCAAGACC GGCCAGATCG CCAACTGTGC 960 GCTGCCCTGC CACAACCCCT TCTTTAGCCA GGATGAGCGC GCCTTCACCG TCTTCTGGAT 1020 CGGCCTGTGG TCGGTGCTCT GCTTCGTCTC CACCTTCGCC ACTGTCTCTA CCTTCCTCAT 1080 CGATATGGAG CGCTTTAAGT ACCCGGAACG GCCCATCATA TTCCTCTCCG CCTGTTACCT 1140 CTTCGTGTCT GTCGGGTACC TGGTGCGCCT GGTGGCAGGA CATGAGAAAG TGGCCTGCAG 1200 CGGCGGCGCT CCGGGTGCTG GCGGACGTGG GGGTGCGGGC GGCGCGGCGG CGGCTGGCGC 1260 AGGGGCAGCG GGACGGGGGG CGAGCAGCCC GGGCGCGCGC GGCGAGTACG AGGAGCTGGG 1320 CGCAGTTGAG CAGCATGTTC GCTATGAGAC CACTGGCCCC GCGCTGTGCA CGGTGGTCTT 1380 TCTCCTTGTC TACTTTTTTG GCATGGCCAG CTCCATCTGG TGGGTAATCC TGTCGCTCAC 1440 GTGGTTCTTG GCAGCTGGCA TGAAGTGGGG TAACGAGGCC ATAGCAGGCT ACTCGCAGTA 1500 CTTCCACCTG GCCGCGTGGC TTGTGCCCAG CGTCAAGTCC ATCGCGGTGC TGGCGCTCAG 1560 CTCCGTAGAC GGCGACCCGG TGGCGGGCAT CTGCTACGTG GGCAACCAGA GCCTTGACAA 1620 CCTACGCGGC TTTGTGCTGG CGCCACTGGT TATCTACCTC TTCATTGGGA CTATGTTTCT 1680 GTTAGCTGGC TTCGTGTCGC TGTTCCGAAT CCGTTCAGTC ATCAAGCAGC AAGGAGGTCC 1740 AACTAAGACA CACAAGCTAG AAAAACTCAT GATCCGCTTG GGCCTCTTCA CCGTGCTCTA 1800 CACGGTGCCC GCTGCCGTCG TTGTCGCCTG CCTTTTCTAT GAGCAGCACA ACCGACCGCG 1860 CTGGGAGGCC ACGCACAACT GCCCATGCCT TCGGGACCTG CAACCGGACC AGGCTCGCAG 1920 GCCCGATTAC GCGGTCTTCA TGCTCAAGTA CTTCATGTGC CTAGTAGTGG GCATCACATC 1980 GGGCGTGTGG GTCTGGTCCG GCAAGACTCT GGAGTCCTGG CGCGCGTTGT GCACTAGGTG 2040 CTGCTGGGCC AGCAAGGGCG CTGCAGTAGG CGCGGGCGCT GGAGGCAGCG GCCCTGGGGG 2100 CAGTGGACCC GGGCCCGGCG GAGGTGGGGG ACACGGCGGA GGCGGGGGAT CCCTCTACAG 2160 CGACGTCAGT ACCGGCCTGA CGTGGCGGTC TGGCACGGCC AGCTCTGTAT CTTACCCTAA 2220 GCAAATGCCA TTGTCCCAGG TCTGAACCCT ACGTGGATGC CCAGAAGGGG CGGAGAGGAG 2280 TGGGGGATGG GGAACCCGTG GGCGGCGAAG GGACCCCAGA CCGGCCAGGG TTCCCACCCC 2340 TTCCCAGTGT TGACTGCTAT AGCATGACAA TGAAGTGTTA ATGGTATCCA TTAGCAGCGG 2400 GGACTTAAAT GACTCCCTTA G 2421 682 amino acids amino acid single linear protein NO NO Mfz8 protein 16 Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu 1 5 10 15 Ala Val Leu Gln Arg Ser Ser Gly Ala Ala Ala Ala Ser Ala Lys Glu 20 25 30 Leu Ala Cys Gln Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly Tyr 35 40 45 Asn Tyr Thr Tyr Met Pro Asn Gln Phe Asn His Asp Thr Gln Asp Glu 50 55 60 Ala Gly Leu Glu Val His Gln Phe Trp Pro Leu Val Glu Ile Gln Cys 65 70 75 80 Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95 Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 105 110 Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gln Tyr Gly Phe Ala 115 120 125 Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gln Gly Asn Pro 130 135 140 Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr Ala Ala 145 150 155 160 Pro Ser Pro Pro Arg Arg Leu Pro Pro Pro Pro Pro Pro Gly Glu Gln 165 170 175 Pro Pro Ser Gly Ser Gly His Ser Arg Pro Pro Gly Ala Arg Pro Pro 180 185 190 His Arg Gly Gly Ser Ser Arg Gly Ser Gly Asp Ala Ala Ala Ala Pro 195 200 205 Pro Ser Arg Gly Gly Lys Ala Arg Pro Pro Gly Gly Gly Ala Ala Pro 210 215 220 Cys Glu Pro Gly Cys Gln Cys Arg Ala Pro Met Val Ser Val Ser Ser 225 230 235 240 Glu Arg His Pro Leu Tyr Asn Arg Val Lys Thr Gly Gln Ile Ala Asn 245 250 255 Cys Ala Leu Pro Cys His Asn Pro Phe Phe Ser Gln Asp Glu Arg Ala 260 265 270 Phe Thr Val Phe Trp Ile Gly Leu Trp Ser Val Leu Cys Phe Val Ser 275 280 285 Thr Phe Ala Thr Val Ser Thr Phe Leu Ile Asp Met Glu Arg Phe Lys 290 295 300 Tyr Pro Glu Arg Pro Ile Ile Phe Leu Ser Ala Cys Tyr Leu Phe Val 305 310 315 320 Ser Val Gly Tyr Leu Val Arg Leu Val Ala Gly His Glu Lys Val Ala 325 330 335 Cys Ser Gly Gly Ala Pro Gly Ala Gly Gly Arg Gly Gly Ala Gly Gly 340 345 350 Ala Ala Ala Ala Gly Ala Gly Ala Ala Gly Arg Gly Ala Ser Ser Pro 355 360 365 Gly Ala Arg Gly Glu Tyr Glu Glu Leu Gly Ala Val Glu Gln His Val 370 375 380 Arg Tyr Glu Thr Thr Gly Pro Ala Leu Cys Thr Val Val Phe Leu Leu 385 390 395 400 Val Tyr Phe Phe Gly Met Ala Ser Ser Ile Trp Trp Val Ile Leu Ser 405 410 415 Leu Thr Trp Phe Leu Ala Ala Gly Met Lys Trp Gly Asn Glu Ala Ile 420 425 430 Ala Gly Tyr Ser Gln Tyr Phe His Leu Ala Ala Trp Leu Val Pro Ser 435 440 445 Val Lys Ser Ile Ala Val Leu Ala Leu Ser Ser Val Asp Gly Asp Pro 450 455 460 Val Ala Gly Ile Cys Tyr Val Gly Asn Gln Ser Leu Asp Asn Leu Arg 465 470 475 480 Gly Phe Val Leu Ala Pro Leu Val Ile Tyr Leu Phe Ile Gly Thr Met 485 490 495 Phe Leu Leu Ala Gly Phe Val Ser Leu Phe Arg Ile Arg Ser Val Ile 500 505 510 Lys Gln Gln Gly Gly Pro Thr Lys Thr His Lys Leu Glu Lys Leu Met 515 520 525 Ile Arg Leu Gly Leu Phe Thr Val Leu Tyr Thr Val Pro Ala Ala Val 530 535 540 Val Val Ala Cys Leu Phe Tyr Glu Gln His Asn Arg Pro Arg Trp Glu 545 550 555 560 Ala Thr His Asn Cys Pro Cys Leu Arg Asp Leu Gln Pro Asp Gln Ala 565 570 575 Arg Arg Pro Asp Tyr Ala Val Phe Met Leu Lys Tyr Phe Met Cys Leu 580 585 590 Val Val Gly Ile Thr Ser Gly Val Trp Val Trp Ser Gly Lys Thr Leu 595 600 605 Glu Ser Trp Arg Ala Leu Cys Thr Arg Cys Cys Trp Ala Ser Lys Gly 610 615 620 Ala Ala Val Gly Ala Gly Ala Gly Gly Ser Gly Pro Gly Gly Ser Gly 625 630 635 640 Pro Gly Pro Gly Gly Gly Gly Gly His Gly Gly Gly Gly Gly Ser Leu 645 650 655 Tyr Ser Asp Val Ser Thr Gly Leu Thr Trp Arg Ser Gly Thr Ala Ser 660 665 670 Ser Val Ser Tyr Pro Lys Gln Met Pro Leu 675 680 6 amino acids amino acid single linear peptide NO NO Amino acid sequence used to design YW157 sense primer 17 Tyr Pro Glu Arg Pro Ile 1 5 5 amino acids amino acid single linear peptide NO NO Amino acid sequence used to design YW158 antisense primer 18 Trp Phe Leu Ala Ala 1 5 

It is claimed:
 1. An isolated nucleic acid molecule encoding a Wnt receptor having an amino acid sequence that is greater than about 90% identical to the amino acid sequence of a Wnt receptor selected from the group consisting of Dfz2, Mfz3, Mfz4, Hfz5, Mfz6, Mfz7, Mfz8, and Cfz1.
 2. A nucleic acid molecule of claim 1 encoding a Wnt receptor having an amino acid sequence that is greater than about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.
 3. A nucleic acid molecule of claim 2 encoding a Wnt receptor having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.
 4. A nucleic acid molecule of claim 3 encoding a Wnt receptor having the amino acid sequence represented as SEQ ID NO:2.
 5. A nucleic acid molecule of claim 1, wherein said molecule encodes a human Wnt receptor.
 6. An isolated Wnt receptor polypeptide having an amino acid sequence that is greater than about 90% identical to the amino acid sequence of a Wnt receptor selected from the group consisting of Dfz2, Mfz3, Mfz4, Hfz5, Mfz6, Mfz7, Mfz8, and Cfz1.
 7. A polypeptide of claim 6, wherein said polypeptide is a human Wnt receptor polypeptide.
 8. A polypeptide of claim 6 having an amino acid sequence that is greater than about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.
 9. A polypeptide of claim 8 having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.
 10. A polypeptide of claim 9 having the amino acid sequence represented as SEQ ID NO:2.
 11. A method of identifying a compound capable of affecting binding of a Wnt polypeptide to a Wnt receptor (WntR) polypeptide, comprising contacting such a WntR with a selected Wnt polypeptide, in the presence and absence of a test compound, measuring the effect of the test compound on the extent of binding between Wnt and said WntR, and identifying said compound as effective to alter binding of a Wnt polypeptide to a WntR polypeptide if its measured effect on the extent of binding is above a threshold level.
 12. The method of claim 11, wherein said threshold is a 2-fold or greater inhibition of binding.
 13. The method of claim 11, wherein said threshold is a 2-fold or greater potentiation of binding.
 14. The method of claim 11, wherein said Wnt polypeptide is wingless (Wg).
 15. The method of claim 11, wherein said WntR polypeptide is Dfz2.
 16. The method of claim 15, wherein said WntR polypeptide has the amino acid sequence represented as SEQ ID NO:2.
 17. The method of claim 11, wherein said test compound is effective to inhibit binding between the Wnt polypeptide and the WntR polypeptide.
 18. The method of claim 11, wherein said test compound is effective to displace the Wnt polypeptide from the WntR polypeptide.
 19. The method of claim 11, wherein said WntR polypeptide is expressed on the surface of a cell transformed with an expression vector encoding said receptor.
 20. The method of claim 19, wherein said cell is a Drosophila Sneider 2 (S2) cell and said expression vector encodes the WntR polypeptide Dfz2.
 21. The method of claim 11, wherein said WntR polypeptide is an N-terminal portion of a full-length WntR polypeptide, said portion including the cysteine-rich amino-terminal domain.
 22. The method of claim 21, wherein said portion is a first part of a fusion protein.
 23. The method of claim 22, wherein said fusion protein further includes a second portion, said second portion containing the constant domain of human IgG.
 24. The method of claim 11, further comprising preparing a pharmaceutical preparation of a compound identified as effective to alter binding of a Wnt polypeptide to a WntR polypeptide. 